Vascular Endothelial Growth Factor mRNA in Pericytes Is Upregulated by Phorbol Myristate Acetate

Yang Kim, Rlffat Y. Imdad, Alan H. Stephenson, Randy S. Sprague, Andrew J. Lomgro

Abstract-Increased mlcrovascular permeability, which occurs m condltlons such as the adult respiratory distress syndrome and diabetes melhtus, IS related to physlcochemlcal alterations m the mlcrovascular barrier We postulate that, m part, capillary pencytes affect mlcrovascular permeablhty via production of a vasoactlve cytokme, VU, vascular endothehal growth factor (VEGF), also known as vascular permeability factor The goal of the present study was to evaluate the effects of phorbol mynstate acetate (PMA), a substance known to produce nonhydrostatlc pulmonary edema m intact ammals, on VEGF gene expression m perlcyte cultures Mlcrovascular pencytes were Isolated from bovme retinas using magnetic mlcrospheres coated with 3G5 monoclonal antibody Perlcyte identity was confirmed both morphologically and by lmmunostammg for a-smooth muscle actm and 3G5 ganghoslde The cultured perlcytes were stimulated with N”-mtro-L-argmme methyl ester (L-NAME, 1 X10e4 mmol/L), anglotensm II (lX30e6 mmol/L), and PMA (5 X lo-’ mmol/L), selected because of then ability to upregulate VEGF mRNA expresslons m other cell types Northern blot analysis was performed usmg [32P]dCTP labeled human VEGF cDNA (Genentech) Lane-loading differences were normalized using mouse GAPDH control cDNA probe VEGF mRNA expression was upregulated by PMA (lo-” to lo-” mol/L) m a dose-dependent manner, whereas neither L-NAME nor anglotensm II affected VEGF mRNA expression m perlcytes These results support the hypothesis that perlcytes mcrease permeability of the endothehal barrier through mcreased VEGF production (Hypertension. 1998;31[part 2]:511-515.)

Key Words: perlcytes n vascular endothehal growth factor w mlcrovascular permeablhty n phorbol mynstate acetate n 3G5 monoclonal antibody

T he regulation of fluid and solute movement across the mlcrovasculature 1s mcompletely described Thus, although

the Starling equation’ allows, with mathematical clarity, a descnptlon of mterrelatlonshlps among those physical forces required for the movement of fluid and small molecules mto and out of exchange vessels, it offers no insight into control mechanisms regulating pressures (hydrostatic and oncotlc) or hydraulic conductance The latter 1s that physical property defining the amount of fluid traversing the vessel wall for a gven pressure difference Therefore, under condmons of constant transbarner hydrostatic and oncotlc pressures, hydrau- hc conductance 1s the preeminent factor m the movement of fluid and solutes across the mlcrovasculature In the mlcrovas- culature, the pnmary determinants of hydraulic conductance have generally been considered to be the degree to which the endothellal intercellular Junctions (the paracellular pathways) are m the “open” state and, to a lesser extent, the state of activity of transcellular pathways 2

Two cells comprise the capillary wall, viz, the endothellal cell and the pencyte 3 The latter has been lmphcated as a regulator of capillary permeabhty pnmanly through its proposed effects on endothehal Intercellular Junctions 3-7 However, the evidence that the pencyte fimchons as a regulator of fluid and solute movement across the rmcrovasculature 1s largely clrcumstanoal 4,8-‘2 The

pencyte of the ludney 1s the glomerular mesangal cell Schlon- dorf?3 has made a strong case for the mesangal cell to function as a controller of glomerular filtration and to partlclpate m the response to local injury by affectmg cell proliferation and base- ment membrane remodelmg, attnbutes that have been ascribed to other nucrovascular pencytes ’ ‘4-‘h Finally, the recent report that mesangal cell? produce VEGF, also known as vascular perme- ab&ty factor,” strengthens the concept that mesangal cells par- ticipate m glomerular filtration and demands an evaluation of the role ofvascular permeabhty factor m pencytes denved from other rmcrovascular beds In prehmmary studies, we have identified the mRNA for vascular permeability factor m bovine retinal pencytes

In our studies of acute lung injury over the past decade, we developed animal models to study the mechamsms of en- hanced nucrovascular permeability One such model used phorbol mynstate acetate (PMA) to produce acute lung injury (nonhydrostatic or permeability inJury) m anesthetized dogs “-” As a necessary first step m testmg the hypothesis that PMA enhanced mlcrovascular permeability via release of vascular permeability factor from mlcrovascular pencytes, m the studies presented here, we evaluated the effects of PMA on the expression of mRNA for vascular permeablhty factor m bovine retmal pencytes

Recewed September 18, 1997, first declsmn October 16, 1997, revuon accepted October 29, 1997 From the Departments of Internal Medlcme and Pharmacolog-rcal and Physmloglcal Suence, Samt LOUIS Unwerslty School of MedIcme, St LOUIS, Mo Correspondence to Andrew J Lomgro, MD, Samt LOUIS Unwerslty, School of Medune 1402 South Grand Blvd, St LOUIS, MO 63104 E-mad

Lonlgro@slu edu 0 1998 Amencan Heart Assoclatlon, Inc

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512 VEGF mRNA Expression and Perlcytes

Selected Abbreviations and Acronyms x1._,: L-NAME = W-mtro-L-argmme methyl ester

PMA = phorbol mynstate acetate VEGF = vascular endotheltal growth factor

Methods

Synthesis of 3G5 Monoclonal Antibody Mouse hybndoma 3G5 cells (Amencan Type Culture Collection) were cultured m a Cellmax-artificial capillary system (Cellco Inc) Hybndoma cells (6X IO’) were maculated mto the extracaplllary space of a moderate pore ~lze artificial capillary module and grown m Dulbecco’s modified Eagle’s mechum with 4 5g/L glucose, 10% fetal bovme serum (Washmgton University, St LOWS, MO), and 1% pemcdhn/streptomycm (Sigma Chemical Co), and maintained at 37’C m a 5% CO, atmosphere The growth medium was replaced when the glucose concentration dropped to 50% of the starting concentration and/or lactate concentrations reached 1 5 to 2 0 g/L Ten days after moculatlon, when the rate of lactate synthesis had increased to 700 to 1000 mg/24 hours, the 3G5 monoclonal antibody was harvested (5 to 10 mL) dally from the extracellular space of the cartridge The harvested me&a were centrifuged, and the supematants were removed, combined, and frozen (-2O“C) before punficatlon of 3G5 IgM using ammomum sulfate precqntatlon and chalysls The yield of 3G5 monoclonal antibody from this hybndoma cell was 3 5 mg/mL using the BCA protem assay (Pierce, Rockford, IL)

Cell Culture Pencytes were isolated from bovine retinas as previously described by Githn and D’Amore*’ and were cultured m mm~mum essential me&urn (Glbco) with 10% fetal bovine serum, 1% perucdhn/strep- tomycm, and 1% amphotencm B (Sigma Chemical Co) m a T-75 flask (Falcon) The cells were Incubated at 37OC m a 5% CO* atmosphere After 24 hours, the me&urn was changed, and thereafter, the medium was changed every other day

After 10 to 14 days m culture, an adchtlonal procedure usmg 3G5 monoclonal antibody’s was introduced to Isolate a pure preparation of pencytes The cells were suspended using 5 mL trypsm/EDTA (Sigma Chemical Co), centrifuged, and resuspended m 10 mL of mechum The cells were mcubated at 4’C for 30 mmutes with Bio-Magnetic bead? (PerSeptwe Biosystems), which had been coated with 3G5 monoclonal antibody AntIbody-coated beads with pencytes attached were isolated magnetically from other cells m the me&urn The pencytes were then freed from the beads m trypsm/EDTA (5 ml), centrifuged, resuspended, and seeded onto P-150 tissue culture dishes Identification of a homogeneous populahon of retmal pencytes was confirmed by then morpholog& features and by fluorescent stammg with anti-ar-smooth muscle actm antibody and the 3G5 monoclonal antibody I8 Perlcytes were used for experiments after 7 to 10 days of mcubation

VEGF mRNA Expression in Bovine Retinal Pericytes Stimulated with Vasoactive Substances Confluent cultures of bovine retinal pencytes grown m P-150 tnsue culture plates were stimulated with L-NAME (1X10m4 mol/L, Sig- maChemlca1 Co), Ang II (lX10m6 mol/L, Sigma Chemical Co), or PMA (5 X lo-’ mol/L, Calbiochem) These concentrations were reported to mcrease VEGF mRNA expression m other cell types 25-27 Pencytes were incubated with these reagents for 3 hours at 37°C m a 5% CO, atmosphere Total RNA was extracted from the stimulated cells, and Northern blot analysis was performed Pencytes m P-150 tissue culture plates incubated with fresh me&urn served as a control

VEGF mRNA Expression in Bovine Retinal Pericytes Stimulated with PMA Confluent cultures of pericytes grown in P-150 tissue culture plates were stimulated mth PMA at 1 X lo-‘, 1 X lo-“, 1 X 1 O-‘, and 1 X lo-’

mol/L, to identify proposed effects on VEGF mRNA expression The stimulated pencytes were mcubated for 3 hours at 37OC m a 5% CO, atmosphere, and total RNA was extracted for Northern blot analysis Pencytes incubated m the same manner without PMA stimulation were used as a control

RNA Isolation and Northern Blot Analysis Total RNA was extracted from mdwidual P-150 tissue culture plates using TRIZOL (Glbco) RNA samples (20 to 25pg/lane) were size-fractionated on a 2% agarose gel contammg 2% formaldehyde and transferred overnight onto Hybond N+ membrane (Amenham) The transferred RNA was cross-hnked to the membrane by ultraviolet machation (Stratahnker) Radioactive probes were synthesized using 25 ng of human VEGF cDNA (a generous gft from Genentech) and [s’P]dCTP (Amersham) with a random pnmed DNA labehng kit (Boehnnger Mannhetm) Bnefly, the membrane was prehybnchzed m a rotatmg hybndlzatlon oven (Boekel) with 50 FL of salmon sperm DNA m 5 mL of Rapid-Hyb buffer (Amersham) at 65°C for 30 mmutes Next, the radiolabeled VEGF cDNA probe was added to the buffer solution and incubated for 2 hours at 55’C After hybnchzanon, the membrane was washed once (at room temperature) m 1 X SSPE solution contammg 0 1% so&urn dodecyl sulfate An addmona wash was performed at 55°C for 30 minutes The final wash was performed with a 0 5% sochum dodecyl sulfate solution at 55OC for 30 minutes Analysis of VEGF mRNA was performed using a Molecular Dynam- KS Computmg PhosphorImager Lane-loadmg chfferences were nor- malized by rehybnchzmg the membrane with racholabeled mouse GAPDH cDNA (Ambion,) probe

Statistical Analysis All experiments were repeated at least three times Results are expressed as the mean-+SE Results were analyzed by an ANOVA for nonpaired data Differences between means were determmed usmg Tukey’s least sign&cant chfference test and P< 05 was considered statistically q&cant

Results

Identification of Bovine Retinal Pericytes Identlfkatlon of bovine retinal pencytes was based on the morphologcal characterlstlcs of the cells and also on the lmmunofluorescence stammg Cultured pencytes are flat, stel- late cells with long, slender processes and short broad filopods ’ The posmve stammg for a-smooth muscle actm mdlcated that the observed cells were not endothehal cells, and the positive stammg for 3G5 ganghoslde indicated that these cells were not fibroblasts nor vascular smooth muscle cells, but conslsted solely of bovine retinal pencytes

Effects of Vasoactive Substances on Vascular Endothelial Growth Factor mRNA Expression To determine whether L-NAME, Ang II, or PMA affects VEGF mRNA expression m pencytes, confluent cultures of pencytes m P-150 tissue culture plates were stimulated and total RNA was extracted for Northern blot analysis (Fig 1) PMA (5X10-* mmol/L) increased the expression of VEGF mRNA 2 420 7 times (P< 05) compared with unstlmulated (control) values (Fig 2) In contrast, concentrations of mRNA for VEGF were not affected by exposure to either L-NAME or Ang II

Pericyte Expression of the mRNA for VEGF in Response to Incremental Doses of PMA The relatlonshlp of VEGF mRNA expression to mcreasmg concentrations of PMA (lo-” mol/L to lo-” mol/L) was

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Figure 1. VEGF mRNA expression of pericytes stimulated by vasoactive substances. Representative Northern blot probed by human VEGF cDNA and mouse GAPDH cDNA, and viewed by the Phosphorlmager. C, control; L-NAME, 1~10~~ mol/L; Ang II, 1 x 1 Om6 mol/L, and PMA, 5x1 Om8 mol/L.

evaluated (Fig 3). All concentrations except for the smallest

(lo-” mol/L), produced rlgmficant increases m VEGF mRNA

expression compared with control values (Fig 4).

Discussion Several pathological conditions, such as diabetes mellitu?’ and

acute lung injury,2”m’x are associated with enhanced microvas-

cular permeability. Although the mechanisms regulating the

movement of fluid across the microvascular barrier are not

comprehensively described, the microvascular cell that has

been implicated as a participant in the regulation of fluid

movement across the microcirculation is the pericyte.“-’ The

pericyte, first described by Rouge? can be thought of as the

“smooth muscle cell” of the capillary. It is abluminal in

location and extends processes down the long axis of, as well as

around, the capillary. Pericytes and endothelial cells are in

intimate contact via adhesion plaques,‘” gap junctions,” and

pericytic processes.“,‘” Pericytes are contractile cells”” and this

property has led to the suggestion that their effects on

400

6

300

E

z 200

k

s 1oc

C

*

*

C L-NAME ANG-II PMA

PcO.05, compared to Control

Figure 2. VEGF mRNA expression in pericytes stimulated with vasoactive reagents. Quantification of multiple experiments (n=3) after normalization to the control signal are shown. Results are expressed as percentage of control VEGF mRNA expression (meaniSE).

*--(iAi’I)II

Figure 3. VEGF mRNA expression of pericytes stimulated by incremental concentrations of PMA. Representative Northern blot probed by human VEGF cDNA and mouse GAPDH cDNA and viewed by the Phosphorlmager.

permeability are mechanical ones related to affecting the

“open” state of endothelial intercellular junctions.‘.“.‘”

The finding that the mesangial cell, the pericyte of the

kidney, produces vascular permeability factor” forced a recon-

sideration of the mechanism(s) of pericyte regulation of fluid

movement across the microvasculature. VEGF is suggested to

alter permeability by enhancing the activity of a transcellular

pathway, recently described as the vesicular-vacuolar or-

ganelle.31 Vascular permeability factor has been reported to be

50 000 times more potent than histamine in its ability to

increase microvascular permeability in several vascular beds.”

We have proposed that the pericyte is the regulator of the

movement of fluid across the microvasculature.” As a first step

in testing this hypothesis, we proposed that PMA, an agent that

produces increased microvascular permeability in intact animal models’fl-2’.“2 enhances microvascular permeability via release

of vascular permeability factor from microvascular pericytes.

In 1995, Aiello et al’” demonstrated the presence of mRNA

for VEGF in several bovine retinal cells including the bovine

retinal pericyte. Moreover, they demonstrated that mRNA for

VEGF is increased under hypoxic conditions and have made a

case for VEGF as a possible participant in retinal neovascular-

6

300

E

E 200

2

s 1 oc

C

*

CON lxlos 1x108 1X10' lxloa

PMA (M)

P<O.O5 compared to Control

Figure 4. VEGF mRNA expression of pericytes stimulated by Incremental concentrations of PMA. Quantification of multiple experiments (n=3) after normalization to the control signal are shown. Results are expressed as percentage of control VEGF mRNA expression (mean?SE). Con, Control.

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514 VEGF mRNA Expression and Pencytes

lzatlon associated with several disease states 33 Shortly thereaf- ter, Takag et al34 reported that the hypoxlc induction of VEGF m retmal pencytes was mehated by adenosme In the study presented here, we isolated, cultured, and punfied bovme retmal pencytes For punficatlon, the use of 3G5 antibody-coated magnehc nucrospheres pernutted the isolahon of pencytes &ee of other rmcrovascular cells The pencytes were ldenhfied morpho- log~ally and by mnnunofluorescent stammg for a-smooth muscle actm and with 3G5 antibody In lmhd expenments, L-NAME and Ang II, which had been reported to mcrease the expression of mRNA for VEGF m other systems, v1z, lung)5 and vascular smooth muscle,3” respectively, as well as PMA were used to stimulate pencytes. Only m the case of PMA was an increase m mRNA for VEGF ldenhfied In subsequent expenmenu, mRNA for VEGF was found to be upregulated m a dose-dependent manner by PMA (lo-” to lo-” mol/L) In the expenments reported here, we did not address the dscrepancy between our results, whch did not show upregulatlon of mRNA for VEGF m the pencytes m response to Ang II, and smular experiments m vascular smooth muscle, which revealed upregulatlon ofVEGF m response to Ang II Bovme rehnd pencytes” as well as human mesangal cells3* have been demonstrated to possess receptors for Ang II In expenments m which pencytes were Isolated and punfied m a manner smnlar to the procedures we used, save for the punficahon step using 3G5 monoclonal anhbodyy3 Ang II was reported to attenuate pencyte relaxahon m response to Increasing the parhal pressure of carbon choxlde m the soluhon bathing the pencytes 39 Thus, one must conclude that either the Ang II receptors have been lost or obscured by use of the 3G5 antibody, that some other factor 1s required for Ang II to function as an agomst for upregulatlon of mRNA for VEGF m the pencyte, or that Ang II is not an agomst for upredahon of

mRNA for VEGF in the pencyte

The present study has not defined the mechamsm whereby PMA regulated VEGF gene expression m pencytes However, PMA IS a protein kmase C agomst,4”-42 which lmphcates a second messenger pathway mvolvmg protein kmase C Indeed the results of both hello et a13” and Takag et al34 are consistent mth that

interpretanon, for both hypo=a43 and adenosme44 affect protein kmase C The findmgs reported here are consistent mth previous reports on signal transduchon pathways of the VEGF gene 4546

In conclusion, the results of our studies show that VEGF mRNA expresslon 1s upregulated by PMA stlmulatlon m pencytes This suggests that pencytes may participate m the increased mlcrovascular permeability m condmons such as the adult respiratory distress syndrome or diabetes melbtus by increasing VEGF synthesis

Acknowledgments This work was supported by an Amencan Heart Assoclatlon Grant (Mlssoun Afflhate) and by Natlonal Institutes of Health (National Heart, Lung and BIood Institute) Grants HL51298 and HL52675 We are indebted to Dr Joseph J Baldassare for gmdmg us m the molecular blologxal techniques used m this work We thank W Jo Schrelwelss for her excellent technical assistance

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Yang Kim, Riffat Y. Imdad, Alan H. Stephenson, Randy S. Sprague and Andrew J. LonigroMyristate Acetate

Vascular Endothelial Growth Factor mRNA in Pericytes Is Upregulated by Phorbol

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1998 American Heart Association, Inc. All rights reserved.

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