Blood pressure is an important factor both in maintaining body homeostasis and in its disruption. Vascular endothelial cells (ECs) are exposed to varying degrees of blood pressure and therefore play an important role in these physiological and pathological events. However, the effect of blood pressure on EC functions remains to be elucidated. In particular, we do not know how ECs sense and respond to changes in hydrostatic pressure even though the hydrostatic pressure is known to affect the EC functions. Here, we hypothesized that the cellular responses, leading to the reported pressure effects, occur at an early stage of pressure exposure and observed the early-stage dynamics in ECs to elucidate mechanisms through which ECs sense and respond to hydrostatic pressure. We found that exposure to hydrostatic pressure causes an early actomyosin-mediated contraction of ECs without a change in cell morphology. This response could be caused by water efflux from the ECs following exposure to hydrostatic pressure. Although only a limited study, these findings do explain a part of the mechanism through which ECs sense and respond to hydrostatic pressure.
Blood pressure plays an essential role in the blood circulatory system helping to supply oxygen and nutrients to every part of the body. Although the body cannot function without blood pressure, chronic and excessive hypertension can also cause various diseases [1,2]. Blood pressure is, therefore, important for the maintenance, as well as the breakdown, of circulatory homeostasis. Blood pressure resulting from the heart's pumping action generates a complicated mechanical condition in the blood vessel. Vascular endothelial cells (ECs) covering the vascular lumen in a monolayer are always exposed to fluid shear stress, cyclic stretching, hydrostatic pressure. ECs are responsible for mediating both the physiological and pathological changes caused by changes in those mechanical stimuli [3,4]. Hence, an understanding of the EC responses to the mechanical stimuli is important to promote health and prevent disease.
Compared with studies examining other hemodynamic stimuli, such as fluid shear stress [5–7] and cyclic stretching [5,8–10], there are few previous studies that have examined the responses of ECs to hydrostatic pressure. The reason for this is that it is unclear how hydrostatic pressure stimulation mechanically acts on ECs. Hydrostatic pressure can promote cellular migration, cell cycle progression, cell proliferation, and apoptosis, depending on the conditions such as the locality, the magnitude of the pressure, and the mode [11–13]. Nevertheless, the knowledge regarding how ECs sense and respond to hydrostatic pressure remains limited. One report has even questioned if pressure can have an effect on ECs .
We recently showed that the hydrostatic pressure promotes cell cycle progression in ECs and leads endothelial cell proliferation without any significant morphological changes being observed . Since these findings were made several hours (3–24 h) after exposure of the ECs to hydrostatic pressure, we still do not understand how ECs sense and respond to pressure from the outset. We hypothesized that the cellular responses that cause the reported pressure effects occur at an early stage of exposure (prior to 3 h). To explore this hypothesis, we have observed and quantified the early-stage dynamics of ECs under different pressure conditions. We focused on morphological changes, intercellular junction formation, cell geometry, actomyosin function, and focal adhesion distribution in ECs. As a result, we conclude that exposure of ECs to hydrostatic pressure causes an actomyosin-mediated endothelial cell contraction without any morphological changes.
Materials and Methods
The mouse monoclonal anti-paxillin antibody (Cat# 610619) was purchased from BD Biosciences (San Jose, CA). The rabbit monoclonal anti-myosin light chain 2 (MLC; Cat# 8505), rabbit polyclonal anti-phospho-myosin light chain 2 (Thr18/Ser19) (phosphorylated MLC (pMLC); Cat# 3674), HRP conjugated anti-mouse IgG antibody (Cat# 7076), and HRP conjugated anti-rabbit IgG (Cat# 7074) were purchased from cell signaling technology (Danvers, MA). Rabbit polyclonal anti-VE-cadherin antibody (Cat# PA5-17401), Alexa Fluor 488-conjugated goat anti-mouse IgG (Cat# A11001), and Alexa Fluor 594-conjugated goat anti-rabbit IgG (Cat# A11012) secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA). Mouse monoclonal anti-VE-cadherin antibody (Cat# sc-9989) was purchased from Santa Cruz Biotechnology (Dallas, TX).
Human umbilical vein endothelial cells (HUVECs; 200-05n, Cell Applications, San Diego, CA) were used from the fifth to eighth passages for the experiments in this study. HUVECs were cultured in 35-mm diameter glass-based dish (3911-035, AGC Techno Glass, Shizuoka, Japan) or plastic dishes (3000-035, AGC Techno Glass), which were precoated with 0.1% bovine gelatin (G9391, Sigma-Aldrich, St. Louis, MO). Medium 199 (M199; 31100-035, Gibco, Thermo Fisher Scientific), containing 20% heat-inactivated fetal bovine serum (FBS; 12438-020, Gibco), 10 μg/L human basic fibroblast growth factor (GF-030-3, Austral Biologicals, San Ramon, CA), and 1% penicillin/streptomycin (P/S; 15140-122, Gibco) was used as a cell culture medium.
Exposure to Hydrostatic Pressure.
Confluent cultures of HUVECs were exposed to a hydrostatic pressure, as previously described  with a slight modification (Fig. 1). Before experiments, the cells were washed twice and incubated with M199 medium containing 10% FBS and 1% P/S (i.e., the experimental medium) for 3 h to wash out any basic fibroblast growth factor present. The pressure system was filled with the experimental medium, and pressure was applied to cells by compressing the volume of the medium. The system was maintained at 37 °C in a CO2 incubator (5% CO2). M199 medium without phenol red (M3769, Sigma-Aldrich) containing 10% FBS, 25 mM HEPES-NaOH, pH 7.4 (H4034, Sigma-Aldrich), 2 mM L-glutamine (25030-081, Thermo Fisher Scientific), and 1% P/S was used for live cell imaging. Cells were exposed to hydrostatic pressures of 50 and 100 mmHg.
For inhibition of actomyosin contractility, HUVECs were incubated with the experimental medium containing 5 μM blebbistatin ((S)-(-)-blebbistatin; B592500, Toronto Research Chemicals, ON, Canada) prepared with DMSO for 30 min before experiments. Blebbistatin inhibits the actomyosin contractility through blocking myosin II-dependent cell processes .
Exposure to Hyperosmotic Shock.
To verify whether the EC responses to hydrostatic pressure were induced by water transfer inside and outside the cells, we observed the dynamics in ECs exposed to hyperosmotic shock, which is capable of forced water transfer from inside to outside of the cells. HUVECs were exposed to hyperosmotic shock by treatment with the experimental medium containing 600 mM glycerol (075-00611, Wako Pure Chemical Industries, Osaka, Japan) for 30 min. The cells were forcibly contracted as a result of the hyperosmotic shock.
Particle Image Velocimetry.
Changes in cell geometry, which are defined as structural changes inside the cell, were assessed using a particle image velocimetry (PIV) analysis of a time lapse series of phase-contrast microscope images by reference to analysis for cell migration velocity using time-lapse imaging [17,18]. Considering the cell itself, as well as its organelles, as the “particle marker,” we applied PIV to the time-series of phase-contrast images of HUVECs captured using an inverted routine microscope (CKX43, Olympus, Tokyo, Japan) with a sCMOS camera (WRAYCAM-SR 130 M, WRAYMER, Osaka, Japan). The PIV analysis was conducted with a PIV plugin [19,20] for ImageJ (U.S. National Institute of Health). In the PIV analysis, a rectangular region of 1272 × 1016 pixels (236 × 188 μm) was divided into small regions of interest (ROIs) of 64 × 64 pixels (11.85 × 11.85 μm). The displacement of each ROI between the images at two time points was calculated based on the cross-correlation function.
After exposure to hydrostatic pressure, the HUVECs were fixed with a 4% paraformaldehyde phosphate buffer solution (163-20145, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). The cells were then permeabilized with 0.1% Triton X-100 (17-1315-01, Pharmacia Biotech, Uppsala, Sweden) in phosphate-buffered saline (PBS) and incubated in 1% Block Ace (BA; UKB40, DS Pharma Biomedical, Osaka, Japan) in PBS to prevent nonspecific antibody adsorption. The cells were then stained using the primary and secondary antibodies diluted in 1% BA in PBS (primary antibodies; 1:200, secondary antibodies; 1:300). Actin filaments were stained using Alexa Fluor 488 conjugated phalloidin (A12379, Thermo Fisher Scientific). Stained HUVECs were observed using an inverted confocal laser-scanning microscope (LSM800, Carl Zeiss, Oberkochen, Germany).
After exposure to hydrostatic pressure, HUVECs were washed with ice-cold PBS containing CaCl2 and MgCl2, scraped into a modified Laemmli buffer (65 mM Tris-HCl [pH 7.5], 0.1 mM EGTA, 0.1 mM EDTA, 1 mM Na3VO4, 1 mM NaH2PO4, 10% glycerol, 2% SDS, 20 mM dithiothreitol, and protease inhibitor cocktail [P8340, Sigma-Aldrich]), and centrifuged at 21,500 × g for 10 min. The supernatant was collected as the whole cell lysate. The lysate samples were separated by SDS-PAGE and then transferred onto an Immun-Blot PVDF membrane (162-0177, Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 1% BA and 0.05% Tween 20 in Tris-buffered saline and then stained using primary and secondary antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution (NKB-101, Toyobo, Osaka, Japan) (primary antibodies; 1:1000, secondary antibodies; 1:5000). The blotted proteins were detected and visualized using clarity western ECL substrate (170-5061, Bio-Rad Laboratories). The molecular weight of each protein was determined based on the migration of Precision Plus Protein Dual Color Standards (161-0374, Bio-Rad Laboratories).
Data Analysis and Statistical Analysis.
For each pressure condition, the experiment was repeated in at least three independent experiments (for PIV analysis and immunoblotting) or three independent experiments (for immunofluorescence staining). Fluorescence images of five arbitrarily determined locations on the glass-based dish were obtained from one experiment: 15 for each condition. Moreover, three cells were randomly selected from each fluorescence image, i.e., a total of 45 cells were used to evaluate each pressure condition.
To measure the overall changes, i.e., morphological changes, in HUVECs subjected to each pressure condition, we monitored the cellular aspect ratio, the orientation angle, and the tortuosity. The outlines of cells selected at random were extracted from the VE-cadherin fluorescence images, and then these parameters were used for a computation based on an ellipse equivalent to the shape of each cell using imagej. The aspect ratio, which has a value ranging from zero (highly elongated) to 1 (a perfect circle), was defined as the ratio between length of the minor and major axes of the ellipsoid. The orientation angle provides the angle between the major axis and a parallel line along the horizontal axis of the original fluorescence image. Tortuosity was defined as the ratio between the cell perimeter and the perimeter of the equivalent ellipse of the cell, increasing from 1 as the shape of the cell becomes more tortuous.
To quantify changes in VE-cadherin structure after exposure to hydrostatic pressure, the ratio of the perimeters of the cellular outline to the inner outline of VE-cadherin was determined by using imagej.
To analyze actomyosin contractility, the colocalization of pMLC with actin filaments was quantitatively obtained using Pearson's correlation coefficient of images processed by zen Imaging Software (Carl Zeiss).
To evaluate the localization of the focal adhesion-associated adaptor protein paxillin, its distribution in each extracted cell was computed with the function “analyze particles” in imagej . For comparison among the variously sized cells, the computed distribution of paxillin was transferred onto a unit circle normalized using an ellipse equivalent to the extracted cell.
Statistical significance was calculated using one-way analysis of variance with post hoc Bonferroni for all parameter (marginally significant: P < 0.1; significant difference: P < 0.05 and P < 0.001).
Morphological Changes in Human Umbilical Vein Endothelial Cells.
We first examined the early-stage morphological responses of HUVECs to hydrostatic pressure (Fig. 2). The HUVECs aspect ratios were distributed over the range 0.3–0.8 under all the pressure conditions examined (Fig. 2(a)). The average aspect ratio was in the range 0.6–0.7, indicating there were no significant differences in aspect ratio over time or at different pressure exposures, although a significant difference in aspect ratio was observed comparing the control HUVECs with HUVECs exposed to 100 mmHg for 30 min. With respect to the orientation angle, the cells exhibited random orientation under all the conditions examined (Fig. 2(b)). The tortuosity was distributed over the range 1.0–1.3, with little differences in the average values and distribution between each of the conditions (Fig. 2(c)). Based on these data, we conclude that during the initial exposure to pressure HUVECs do not undergo any remarkable morphological changes
Intercellular Junction Formation Following Exposure to Pressure.
Given the lack of changes in cell morphology seen following exposure of HUVECs to pressure, we next observed the expression of VE-cadherin, as well as the formation of VE-cadherin containing intercellular junctions (Fig. 3). VE-cadherin can act as a mechano-sensor for mechanical stimuli . The total expression levels of VE-cadherin were not affected by exposure to hydrostatic pressure even up to 3 h of exposure (Fig. 3(a)). After 30 min of pressure exposure, VE-cadherin was seen to be present in a wedge-like structure present between neighboring cells (Fig. 3(b)). This structure was quantitatively evaluated based on the ratio of the cellular outline to inner outline of VE-cadherin (Fig. 3(c)). This structure was still visible after 3 h of 50 mmHg hydrostatic pressure, while the structure under 100 mmHg pressure was clearly less visible after 1 h of exposure. There is a difference in the duration time to sustain the changes in VE-cadherin structure depending on the pressured conditions. These changes in VE-cadherin intercellular junctions suggest that HUVECs contract in response to exposure to hydrostatic pressure.
Changes in Cellular Geometry.
To further evaluate the contraction of HUVECs caused by pressure exposure, we next investigated cellular dynamics, i.e., changes in cell geometry, under the different pressure conditions. Cellular dynamics occurring during the first 30 min of exposure to hydrostatic pressure were measured using PIV (Fig. 4). HUVECs tended to round up their geometries toward their middle following exposure to the different pressure conditions, whereas control HUVECs migrated in a definite direction or exhibited a rotational movement (Fig. 4(a)). These dynamics in the cell geometries were clearly seen as early as 5 min after pressure exposure (Fig. 4(b)). Although the cellular dynamics were different between control HUVECs and HUVECs exposed to the different pressure conditions, the averaged displacement of the cell geometry did not show any significant differences (Fig. 4(c)), except that it shows marginally significant between two pressured conditions from 0 to 5 min after exposure. As a result of this PIV analysis, we conclude that HUVEC cells contract following exposure to pressure.
The conclusion from the PIV analysis that HUVECs contract following exposure to pressure was also supported by an assessment of actomyosin contractility (Fig. 5). Exposure of HUVECs to hydrostatic pressure increased the phosphorylation level of MLC, which plays an important role in the contractile machinery of cells  (Fig. 5(a)). Actin filaments developed and formed actin stress fibers after exposure to pressure. In addition, pMLC found to be colocalized with the actin stress fibers that developed in the cell periphery following exposure to pressure (Fig. 5(b)). The degree of colocalization (assessed using Pearson's correlation coefficient) of pMLC with actin filaments decreased slightly from 30 min to 3 h of exposure to hydrostatic pressure (Fig. 5(c)). Under control conditions, no significant colocalization of pMLC with actin filaments was observed, and there was no evidence for the formation of actin filaments. These data thus demonstrate that exposure to hydrostatic pressure causes actomyosin-mediated cell contraction during the early stages of pressure exposure.
Focal Adhesion Distribution.
Actomyosin contractility is known to regulate the molecular kinetics of focal adhesions . Therefore, we the examined the expression and distribution of paxillin, which is a focal adhesion-associated adaptor protein. The total expression levels of paxillin in HUVECs were not changed by any of the pressure condition tested up to 3 h (Fig. 6(a)). Particulate paxillin staining was clearly visible in the cells following exposure to pressure and increased with time (Fig. 6(b)). The area of paxillin increased with the time of exposure to pressure (Fig. 6(c)). Most of the paxillin was expressed at the cell periphery, and the paxillin found there did not change its distribution following exposure to pressure (Fig. 6(d)). These results indicate paxillin increased its area at the cell periphery in order to support the development of the actin stress fibers that are responsible for cell contraction.
Importance of Actomyosin Contractility.
To verify the importance of actomyosin contractility for EC responses to hydrostatic pressure, we inhibited the actomyosin contractility due to hydrostatic pressure exposure by treatment of 5 μM blebbistatin for 30 min. The absences of phosphorylation of MLC (Fig. 7(a)), development of actin stress fibers (Fig. 7(b)), and colocalization of pMLC with actin filaments (Figs. 7(b) and 7(c)) showed the inhibition of actomyosin contractility in HUVECs by treatment of the blebbistatin. Changes in VE-cadherin junction formation (Figs. 7(e) and 7(f)) as well as expression of paxillin with large area (Figs. 7(h) and 7(i)), which were induced by the pressure exposure, were not observed though treatment of blebbistatin has not affected expression of VE-cadherin (Fig. 7(d)) and distribution of paxillin (Figs. 7(g) and 7(j)). Consequently, those results suggest the actomyosin contractility of ECs plays an important role for the EC responses to hydrostatic pressure.
Intercellular Junction Formation Under Hyperosmotic Shock.
To investigate what causes actomyosin-mediated EC contraction following exposure to hydrostatic pressure, we observed the formation of VE-cadherin intercellular junction and actomyosin contractility when the cells were forced to contract by hyperosmotic shock (Fig. 8). After 30 min of osmotic pressure exposure, VE-cadherin formed wedge-like structures (Fig. 8(a)), which were similar to those seen following exposure to hydrostatic pressure. In addition, actomyosin contractility was also observed after exposure to osmotic pressure (Fig. 8(b)). These effects are caused by cell contraction following water efflux from the cells exposed to hyperosmotic shock, suggesting that water efflux may also occur in cells exposed to hydrostatic pressure.
In this study, exposure of HUVECs to hydrostatic pressure caused an actomyosin-mediated cell contraction without any remarkable changes in morphology. As was observed in an earlier study , exposure to hydrostatic pressure did not affect the aspect ratio, orientation angle, and tortuosity, changes in which indicate morphological changes in endothelial cells (Fig. 2). In contrast, following exposure to hydrostatic pressure the geometry of the cells rounded up toward their middle (Fig. 4). This result suggests that exposure to hydrostatic pressure induces cell contraction. This notion is supported by the following findings; (1) After 1 h of exposure, VE-cadherin was found to be present in wedge-like structures between neighboring cells (Fig. 3); (2) exposure to hydrostatic pressure induced the development of actin stress fibers at the cell periphery and actomyosin contractility (Fig. 5); (3) after exposure to hydrostatic pressure, large areas of paxillin were formed to support the ability of the newly developed actin stress fiber to generate a contractile force (Fig. 6); (4) similar wedge-like VE-cadherin containing structures were observed after the forced cell contraction caused by hyperosmotic shock (Fig. 8); (5) Absence of those changes in HUVECs in response to hydrostatic pressure was exhibited by inhibition of the actomyosin contractility (Fig. 7).
The parameters that normally define cellular morphological changes were not changed following exposure to hydrostatic pressure over the short term of this study (i.e., up to 3 h) (Fig. 2). Exposure to hydrostatic pressure has also been shown to not affect the morphology of HUVECs over longer exposure times (3–24 h) . Similarly, bovine aortic ECs did not show any morphological changes in response pressure exposure over the medium and long term (3 h, 24 h, and 7 days) . The results of this study are therefore in agreement with these reports. On the other hand, in several other studies, endothelial morphology, including elongation, changes in actin distribution, or the appearance of multilayers, has been reported to be affected by exposure to pressure [25–27]. The discrepancies between these studies and ours might arise due to differences in the system used to apply pressure or in the cell type. In this regard, different morphological responses in human and bovine ECs have been reported [15,25].
In our study, exposure to hydrostatic pressure induced actomyosin contractility, resulting in changes in VE-cadherin formation and the redistribution of paxillin (Fig. 3–6). VE-cadherin is known to act as a tension sensor . Mechano-transduction by VE-cadherin complexes triggers cytoskeletal remodeling and also activates signals that alter intercellular junctions with the involvement of an actomyosin contractile force . The actomyosin contractile force generates a tension that is exerted on VE-cadherin leading to its disruption [28,29]. This disrupted VE-cadherin caused by mechanical stimuli, such as fluid shear stress , appears as a wedge-like structure that was also observed after exposure of HUVECs to hydrostatic pressure in this study (Fig. 3). Actomyosin-mediated tension is necessary for the maturation of focal adhesions [30,31]. Fluid shear stress  or cyclic stretching  induces an increase in the area of paxillin with development of actin stress fibers. We also observed the increase in the area of paxillin staining around the cell periphery under the pressured condition (Fig. 6), where the phosphorylation of pMLC (Fig. 5), i.e., actomyosin contractility, was also observed. Inhibition of actomyosin contractility exhibited absences of those changes in HUVECs in response to hydrostatic pressure (Fig. 7). Consequently, we conclude that hydrostatic pressure causes actomyosin-mediated endothelial cell contraction.
The maximum pressure that the HUVECs in this study were exposed to was 100 mmHg. This pressure level is not sufficient to alter or damage the structure of intracellular proteins directly . Therefore, the actomyosin contractility that caused the cell contraction is unlikely to be directly induced by such a level of hydrostatic pressure. One clue to answer the question of how exposure to hydrostatic pressure causes the contraction of HUVECs lies in the data we obtained when we observed a disruption of VE-cadherin due to the forcible cell contraction caused by hyperosmotic shock (Fig. 8). Hyperosmotic shock induces water efflux from the inside of cells, resulting in cell contraction. This phenomenon is supported by a previously described kinetic model , in which water flux is defined by the difference between the hydrostatic and osmotic pressures across the cell membrane. Here, we hypothesize that hydrostatic pressure also causes water efflux from the cells as is the case with hyperosmotic shock. Cell is a gel-like substance covered with the cell membrane composed of a hydrophobic lipid bilayer. Large amount of water is contained in the cell with a cytoskeleton and various intracellular organelles. Thus, changes in the extracellular hydrostatic pressure cause a hydrostatic pressure gradient, albeit only for a short time, to balance the intracellular and extracellular pressures. Our experimental system applied hydrostatic pressure to the HUVECs by compressing the experimental medium (Fig. 1). Given that the compressibility of the experimental medium is approximately that of water (4.43 × 10−10 Pa−1 at 37 °C ), the volume compression of the medium required to produce the predetermined pressure values of 50 and 100 mmHg is very small. Therefore, under the conditions used in this study, since the osmotic pressure does not change, the water efflux from the cell is defined only by changes in the hydrostatic pressure. Based on the aforementioned kinetic model (Fig. 9), we conclude that exposure to hydrostatic pressure might cause water efflux from the cells resulting in actomyosin-mediated cell contraction.
This study shows vascular endothelial responses to hydrostatic pressure in the early stage of exposure. Not only blood vessel but also many various organs, for example, the bladder or retina, are physiologically exposed to hydrostatic pressure. Several researches have been conducted focusing on those organs. Rat bladder urothelial cells release adenosine triphosphate when exposed to sustained hydrostatic pressure in the physiological threshold range . The pressure-induced amplification of adenosine triphosphate signal in rat urothelial cells activates P2X4 receptors, which mediate activation of the caspase-1 inflammatory response . For the retinal pigment epithelial cells, tight junction protein occludin regulates tight junction permeability in response to changes in hydrostatic pressure . However, knowledge is still lacking for a complete understanding of mechanisms through which cells sense and respond to hydrostatic pressure.
This study provides new insights into how ECs respond to hydrostatic pressure based on an analysis of early-stage dynamics under different pressure conditions. However, we could not determine the mechanism by which ECs convert hydrostatic pressure to intracellular biochemical signals, which induce actomyosin-mediated cell contraction. In addition, we could not directly observe water transfer from the inside to the outside of the cell. Although information about the water transfer is still limited, aquaporin, which is a selective water channel, probably plays an important role in the system for the water transfer. By solving these problems in the future, we believe it will be possible to understand how ECs sense and respond to hydrostatic pressure in more detail. We investigated early-stage dynamics in ECs under the static pressure condition in this study; ECs are subjected to pulsatile blood pressure under the physiological condition. The pulsatile blood pressure reportedly enhances EC proliferation . Thus, there is also the necessity of future work about the effects of the pulsatile pressure on the EC responses.
Here, we investigated the cellular responses to hydrostatic pressure in the early stages of exposure. We demonstrated that hydrostatic pressure causes actomyosin-mediated EC contraction in the absence of morphological changes. These responses can be induced by water efflux from the HUVECs that arises as a result of the hydrostatic pressure stimulation. Although limited in scope, this study does in part explain the mechanism through which ECs sense and respond to pressure, which has been a long-standing question in the field.
This work was supported in part by Building of Consortia for the Development of Human Resources in Science and Technology from the Japan Science and Technology Agency (to D.Y.).
During his time at the University of Houston (1983-1984), Masaaki Sato (M.S.) was honored to be able to attend “Symposium on Frontiers in Applied Mechanics and Biomechanics” held at UCSD, July 21–23, 1984. This symposium was planned to celebrate Professor Fung's 65th birthday. As a result of studying the biomechanics of vascular walls since 1971, M.S. was very familiar with Professor Fung's excellent work and reputation in the field of biomechanics. M.S. was very grateful to have the opportunity to meet Professor Fung at the UCSD symposium and remembers clearly being very impressed with him. Since that first meeting, M.S. was always happy to meet him at many international conferences and meetings they jointly attended. Dr. H. Abé (a former president of Tohoku University) and M.S. organized the 5th Japan-USA-Singapore-China Conference of Biomechanics, in Sendai, just after the 3rd WCB in Sapporo, Japan, 1998. In particular, this conference made a great impression on M.S. because Professor Fung and his wife were able to attend and spent the whole period with M.S. and Dr. H. Abé. M.S. was again invited to attend the International Symposium on Genomic Biomechanics: Frontier of the 21st Century held at UCSD in 2008 to celebrate Dr. Fung's 90th birthday. We look forward to celebrating his 100th birthday and wish him continued good health and longevity.