Development of a Novel Culture System : To Investigate the Effect of Hydrostatic Pressure
on Renin‑Angiotensin Sys tem for Monocyte
Takehiko KUNIEDA,Yoshinobu MANOME,Mariko N AKAMURA, Hiroaki YOSHIDA,Tetsuya KAWAMURA,and Tsuneya O HNO
Division of Nephrology and Hypertension,Department of Internal Medicine, The Jikei University School of Medicine
Microbiology,The Jikei University School of Medicine
Division of Pediatric Nephrology,Vanderbilt University Medical Center
ABSTRACT
Hemodynamic forces,whi ch include hydrostatic pressure,stretch,and shear stress,have been shown to be important regulators of vascular struct ure and function. Although effects of vascular wall stretching and shear stress have been profoundl y studied,very little is known about the effects of hydrostatic pressure on monocytic response. We reasoned that hydrostatic pressure may be the most significant physiological regulator of monocyt ic function. To examine the direct effect of hydrostatic pressure on cells,we have developed a new in vitro culture device:“Versatile Culture System (VCS)”. VCS consists of culture chamber ,a control valve,and a pressure sensor through which media pumped to a waste at a predetermi ned rate. We design it for a wide variety of cell culture and it has ability to load pressure ranging from 0‑760 mmHg.
In this system,monocytic cell line THP1 were grown in the suspension cell chamber which was specially designed for suspension cell culture wi thout being affected by shear stress. To clarify whether pure hydrostatic pressure itself affects the renin‑angiotensin system (RAS),which has been modulated by stimulus of hemodynamic overload,we exami ned the expression of angiotensin
‑converting enzyme(ACE)and angiotensin type 1 receptor(AT1)genes of cultured monocytic cells. We subjected THP1 to 100 mmHg or 200 mmHg constant pressure for up to 12 hours. Quantitative RT‑PCR analysis revealed ACE and AT1 mRNA have a t endency of alterations in a different manner by exposure to hydrostatic pressure. The present study suggest that physiological hydros- tatic pressure has an ability to modulate RAS of monocytic cells at different magnitude of pressure.
We conclude that VCS provides a mechanistic insight into the role of hydrostatic pressure on various cell functions. (Jikeikai Med J 2002;49:13‑21)
Key words:culture system,pressure,monocyte,angiotensin‑converting enzyme,angiotensin type 1 receptor
I NTRODUCTION
The hemodynamic environment to which blood vessel is exposed is known t o regulate its structure during development. Recent wor k in cellular and
molecular biology has uncovered a set of complex interactions between various components of the blood vessel including endothelium,s mooth muscle,matrix and circulating blood cel ls such as leukocytes (monocytes,lymphocytes,and polymorphonuclear leu-
Received for publication,December 3,2001
国枝 武彦,馬目 佳信,中村真理子,吉田 裕明,川村 哲也,大野 典也
Mailing address:Takehiko K UNIEDA,Division of Nephrology and Hypertension,Department of Internal Medicine,3‑25‑8,Nishi‑ Shimbashi,Minato‑ku,Tokyo 105‑8461,Japan.
13
roundings.
Hypertension is characterized by structural alter- ations of the vasculature,depending on the size and function of the particular vessel. A large number of growth factors,cytokines,and vas oregulatory mole- cules have been proposed to explain the structural alterations that occur dur ing hypertension. The renin‑angiotensin system ( RAS)is an important regu-
lator of vascular tone and sodium‑electrolyte balance, and its principal mediator,angiotensin II(Ang II), may also be important as a trophic factor for the maintenance of vascular s tructure. For example,
Ang II has been extensively implicated in the develop- ment of c a r d i a c h y p e r t r o p h y a s s o c i a t e d w i t h hemodynamic overload,hyper tension,and myocar- dial infarction. The components of the RAS are present in many tissues and there is evidence for local de novo byosynthesis of Ang II. These tissue‑based systems can be regulated i ndependently of the endo- crine RAS,and it has been suggested that the plasma RAS is predominantly impor tant for acute regulatory mechanisms whereas the l ocal RAS may be more involved in chronic aspect s of cardiovascular regula-
tion ,and considered as an important autocrine‑par- acrine system for the regulation of vascular tone and structure . Several in vi vo studies have demonstrat- ed that mRNA expression of ACE ,and AT1 are upregulated in response to pressure overload or after myocardial infarction in var ious animal species. It has also been shown that s hear stress mechanisms of ACE regulation may play an i mportant role in the control of the local RAS f or changes in vascular structure . The effect of shear stress or stretch on the release of various vasoact ive substances has been
pressure‑loading apparatus and reported that whether hydrostatic pressure modul ates local RAS genes,in- cluding ACE and AT1 gene expression in human monocytic cells.
MATERIAL AND METHODS Pressure‑loading appar atus
A new in vi tro culture device;“Versatile Culture System (VCS)”,have devel oped to clarify the effects of hemodynamic force on bot h vascular wall and circulating cells. This enabl ed us to culture a wide variety of cell with the change of cell chamber and to load pressure ranging f rom 0‑760 mmHg. Fig.1 shows schematic diagram of t he VCS designed to suspension cell culture wi th constant pressure. Cul- ture medium was pumped to a waste through culture chamber,pressure sensor and a cont rol valve at a predetermined flow. All s ystem was set up in a standard CO incubator(As tec,Fukuoka,Japan)and the entire period of perfus ion was recorded on chart using a printer(M & S,Tokyo,Japan;LC ‑1001).
We have modified a disposable fuser(Terumo,Tokyo, Japan;L060K01)as suspension cell chamber and used syringe pump(Terumo,Tokyo,Japan;TE ‑311)and Swagelok valve (Nupro,Ohi o,U.S.A.;SS‑SS2)to regulate pressures. The ot her disposable compo-
nents such as connecting tube,pressure sensor(PR
‑AS123S)and syringe were purchased from Terumo (Tokyo,Japan).
Cell culture
THP1 ( American Type Culture Collection),a
human monocytic cell line,was grown in culture using RPMI1640 (GIBCO‑BRL) ,supplemented with 10%
fetal bovine serum. Cells were resuspended in 175 cm culture flask(Falcon)and passaged at 1:10 in a 95% humidified air/5% CO envi ronment at 37°C.
Initially,30 ml of cell suspension at density of 1×10 cells/ml were incubated for 12 hours to eliminate possible effects due to the addition of fresh medium.
Then,cells were transferred to a chamber at 5×10 cells/ml(chamber volume 66 ml). All cells used were between passages 6 and 12 from the initial purchase.
To note,extremely care was taken to avoid the load- ing of additional mechanical stress to cells(e.g.,con- ventional centrifugation for subculture).
Mechanical stimulation
In this s tudy,culture medium,which was prein- cubated 6 hours in CO incubator,was pumped at 4 ml/hr and either 100±2 or 200±2 mmHg of constant pressure,achieved by adj usting the control valve.
The cells were exposed to pressure for 6 and 12 hours and compared to the control specimens from the same set of replicate cultures under 0 mmHg These parallel sets of experiments in each time course were perfor- med simultaneously. The partial pressure of oxygen and pH of the medium showed no significant changes for 24 hours after the appl ication of pressure and the viability of cells was ascer tained by a trypan blue exclusion assay.
RNA isolation and quantitative PCR
Total cellular RNA i n monocytic cell lines were isolated by RNeasy Mini Ki t(Qiagen)at 0,6,12 hours of pressure‑loading,and t otal RNA was prepared according to the manufact urerʼs recommendations. The RNA was dissolved in 30μl of H O and the concentration was determi ned by spectrophotometer (A 260/280 nm). Reverse transcription of RNA was carried out in 20μl of a r eaction mixture by using Omniscript(Qiagen),cont aining 1μg of total RNA.
The reaction master mix was prepared according to the manufacturerʼs protocol to give final concentra- tions of 1×reaction buffer,0.5 mM each dNTPs,0.5 units/μl RNase inhibitor,0. 2 units/μl reverse tran- scriptase,0.5μM random hexamers. To monitor gene expression we used r eal time PCR analysis, which has been described previously . Briefly,50 ng of total RT product was added to a 50μl of a reaction mixture. PCR amplificat ion was carried out in the presence of 1×reaction buf fer,0.2 mM each dNTPs, 3.5 mM MgCl,0.02 units/μl AmpliTaq Gold DNA polymerase,a 300 nM concent ration of the primers, and a 200 nM concentration of the corresponding probe. Primers and probes of ACE,AT1,GAPDH genes were designed by the Primer Express Program according to Heid et al. For sequence information of all oligonucleotides and pr obes,see Table 1. All primers were synthesized at Lifetech Oriental(Tokyo, Japan)using conventional nucleic acid synthesis chem- A Novel In Vitro Culture System
Fig.1. Versatile Culture System (VCS)
Unit for constant pressure/suspension cell culture