(Hypertension. 1998;31:511.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Vascular Endothelial Growth Factor mRNA in Pericytes Is Upregulated by Phorbol Myristate Acetate
From the Departments of Internal Medicine and Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO.
Correspondence to Andrew J. Lonigro, MD, Saint Louis University, School of Medicine. 1402 South Grand Blvd, St. Louis, MO 63104. E-mail Lonigro{at}slu.edu
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Abstract |
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Increased microvascular permeability, which occurs in conditions such as the adult respiratory distress syndrome and diabetes mellitus, is related to physicochemical alterations in the microvascular barrier. We postulate that, in part, capillary pericytes affect microvascular permeability via production of a vasoactive cytokine, viz, vascular endothelial growth factor (VEGF), also known as vascular permeability factor. The goal of the present study was to evaluate the effects of phorbol myristate acetate (PMA), a substance known to produce nonhydrostatic pulmonary edema in intact animals, on VEGF gene expression in pericyte cultures. Microvascular pericytes were isolated from bovine retinas using magnetic microspheres coated with 3G5 monoclonal antibody. Pericyte identity was confirmed both morphologically and by immunostaining for
-smooth muscle actin and 3G5 ganglioside. The cultured pericytes were stimulated with N
-nitro-L-arginine methyl ester (L-NAME, 1x10-4 mmol/L), angiotensin II (1x10-6 mmol/L), and PMA (5x10-8 mmol/L), selected because of their ability to upregulate VEGF mRNA expressions in other cell types. Northern blot analysis was performed using [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 (10-9 to 10-6 mol/L) in a dose-dependent manner, whereas neither L-NAME nor angiotensin II affected VEGF mRNA expression in pericytes. These results support the hypothesis that pericytes increase permeability of the endothelial barrier through increased VEGF production.
Key Words: pericytes • vascular endothelial growth factor • microvascular permeability • phorbol myristate acetate • 3G5 monoclonal antibody
Abbreviations: Ang II = angiotensin II • L-NAME = N-nitro-L-arginine methyl ester • PMA = phorbol myristate acetate • VEGF = vascular endothelial growth factor
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Introduction |
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The regulation of fluid and solute movement across the microvasculature is incompletely described. Thus, although the Starling equation1 allows, with mathematical clarity, a description of interrelationships among those physical forces required for the movement of fluid and small molecules into and out of exchange vessels, it offers no insight into control mechanisms regulating pressures (hydrostatic and oncotic) or hydraulic conductance. The latter is that physical property defining the amount of fluid traversing the vessel wall for a given pressure difference. Therefore, under conditions of constant transbarrier hydrostatic and oncotic pressures, hydraulic conductance is the preeminent factor in the movement of fluid and solutes across the microvasculature. In the microvasculature, the primary determinants of hydraulic conductance have generally been considered to be the degree to which the endothelial intercellular junctions (the paracellular pathways) are in the "open" state and, to a lesser extent, the state of activity of transcellular pathways.2
Two cells comprise the capillary wall, viz, the endothelial cell and the pericyte.3 The latter has been implicated as a regulator of capillary permeability primarily through its proposed effects on endothelial intercellular junctions.3–7 However, the evidence that the pericyte functions as a regulator of fluid and solute movement across the microvasculature is largely circumstantial.4,8–12 The pericyte of the kidney is the glomerular mesangial cell. Schlon-dorff13 has made a strong case for the mesangial cell to function as a controller of glomerular filtration and to participate in the response to local injury by affecting cell proliferation and basement membrane remodeling, attributes that have been ascribed to other microvascular pericytes.5,14–16 Finally, the recent report that mesangial cells produce VEGF, also known as vascular permeability factor,17 strengthens the concept that mesangial cells participate in glomerular filtration and demands an evaluation of the role of vascular permeability factor in pericytes derived from other microvascular beds. In preliminary studies, we have identified the mRNA for vascular permeability factor in bovine retinal pericytes.
In our studies of acute lung injury over the past decade, we developed animal models to study the mechanisms of enhanced microvascular permeability. One such model used phorbol myristate acetate (PMA) to produce acute lung injury (nonhydrostatic or permeability injury) in anesthetized dogs.18–21 As a necessary first step in testing the hypothesis that PMA enhanced microvascular permeability via release of vascular permeability factor from microvascular pericytes, in the studies presented here, we evaluated the effects of PMA on the expression of mRNA for vascular permeability factor in bovine retinal pericytes.
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Methods |
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Synthesis of 3G5 Monoclonal Antibody
Mouse hybridoma 3G5 cells (American Type Culture Collection) were cultured in a Cellmax-artificial capillary system (Cellco Inc). Hybridoma cells (6x107) were inoculated into the extracapillary space of a moderate pore size artificial capillary module and grown in Dulbecco’s modified Eagle’s medium with 4.5g/L glucose, 10% fetal bovine serum (Washington University, St. Louis, Mo), and 1% penicillin/streptomycin (Sigma Chemical Co), and maintained at 37°C in a 5% CO2 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 inoculation, 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) daily from the extracellular space of the cartridge. The harvested media were centrifuged, and the supernatants were removed, combined, and frozen (-20°C) before purification of 3G5 IgM using ammonium sulfate precipitation and dialysis. The yield of 3G5 monoclonal antibody from this hybridoma cell was 3.5 mg/mL using the BCA protein assay (Pierce, Rockford, IL).
Cell Culture
Pericytes were isolated from bovine retinas as previously described by Gitlin and D’Amore22 and were cultured in minimum essential medium (Gibco) with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% amphotericin B (Sigma Chemical Co) in a T-75 flask (Falcon). The cells were incubated at 37°C in a 5% CO2 atmosphere. After 24 hours, the medium was changed, and thereafter, the medium was changed every other day.
After 10 to 14 days in culture, an additional procedure using 3G5 monoclonal antibody23 was introduced to isolate a pure preparation of pericytes. The cells were suspended using 5 mL trypsin/EDTA (Sigma Chemical Co), centrifuged, and resuspended in 10 mL of medium. The cells were incubated at 4°C for 30 minutes with Bio-Magnetic beads24 (PerSeptive Biosystems), which had been coated with 3G5 monoclonal antibody. Antibody-coated beads with pericytes attached were isolated magnetically from other cells in the medium. The pericytes were then freed from the beads in trypsin/EDTA (5 ml), centrifuged, resuspended, and seeded onto P-150 tissue culture dishes. Identification of a homogeneous population of retinal pericytes was confirmed by their morphological features and by fluorescent staining with anti--smooth muscle actin antibody and the 3G5 monoclonal antibody.18 Pericytes were used for experiments after 7 to 10 days of incubation.
VEGF mRNA Expression in Bovine Retinal Pericytes Stimulated with Vasoactive Substances
Confluent cultures of bovine retinal pericytes grown in P-150 tissue culture plates were stimulated with L-NAME (1x10-4 mol/L; SigmaChemical Co), Ang II (1x10-6 mol/L; Sigma Chemical Co), or PMA (5x10-8 mol/L; Calbiochem). These concentrations were reported to increase VEGF mRNA expression in other cell types.25–27 Pericytes were incubated with these reagents for 3 hours at 37°C in a 5% CO2 atmosphere. Total RNA was extracted from the stimulated cells, and Northern blot analysis was performed. Pericytes in P-150 tissue culture plates incubated with fresh medium 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 with PMA at 1x10-9, 1x10-8, 1x10-7, and 1x10-6 mol/L, to identify proposed effects on VEGF mRNA expression. The stimulated pericytes were incubated for 3 hours at 37°C in a 5% CO2 atmosphere, and total RNA was extracted for Northern blot analysis. Pericytes incubated in the same manner without PMA stimulation were used as a control.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from individual P-150 tissue culture plates using TRIZOL (Gibco). RNA samples (20 to 25µg/lane) were size-fractionated on a 2% agarose gel containing 2% formaldehyde and transferred overnight onto Hybond N+ membrane (Amersham). The transferred RNA was cross-linked to the membrane by ultraviolet irradiation (Stratalinker). Radioactive probes were synthesized using 25 ng of human VEGF cDNA (a generous gift from Genentech) and [32P]dCTP (Amersham) with a random primed DNA labeling kit (Boehringer Mannheim). Briefly, the membrane was prehybridized in a rotating hybridization oven (Boekel) with 50 µL of salmon sperm DNA in 5 mL of Rapid-Hyb buffer (Amersham) at 65°C for 30 minutes. Next, the radiolabeled VEGF cDNA probe was added to the buffer solution and incubated for 2 hours at 55°C. After hybridization, the membrane was washed once (at room temperature) in 1x SSPE solution containing 0.1% sodium dodecyl sulfate. An additional wash was performed at 55°C for 30 minutes. The final wash was performed with a 0.5% sodium dodecyl sulfate solution at 55°C for 30 minutes. Analysis of VEGF mRNA was performed using a Molecular Dynamics Computing PhosphorImager. Lane-loading differences were normalized by rehybridizing the membrane with radiolabeled 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 determined using Tukey’s least significant difference test and P<.05 was considered statistically significant.
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Results |
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Identification of Bovine Retinal Pericytes
Identification of bovine retinal pericytes was based on the morphological characteristics of the cells and also on the immunofluorescence staining. Cultured pericytes are flat, stellate cells with long, slender processes and short broad filopods.5 The positive staining for
-smooth muscle actin indicated that the observed cells were not endothelial cells, and the positive staining for 3G5 ganglioside indicated that these cells were not fibroblasts nor vascular smooth muscle cells, but consisted solely of bovine retinal pericytes.
Effects of Vasoactive Substances on Vascular Endothelial Growth Factor mRNA Expression
To determine whether L-NAME, Ang II, or PMA affects VEGF mRNA expression in pericytes, confluent cultures of pericytes in P-150 tissue culture plates were stimulated and total RNA was extracted for Northern blot analysis (Fig 1). PMA (5x10-8 mmol/L) increased the expression of VEGF mRNA 2.4±0.7 times (P<.05) compared with unstimulated (control) values (Fig 2). In contrast, concentrations of mRNA for VEGF were not affected by exposure to either L-NAME or Ang II.
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Pericyte Expression of the mRNA for VEGF in Response to Incremental Doses of PMA
The relationship of VEGF mRNA expression to increasing concentrations of PMA (10-9 mol/L to 10-6 mol/L) was evaluated (Fig 3). All concentrations except for the smallest (10-9 mol/L), produced significant increases in VEGF mRNA expression compared with control values (Fig 4).
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Discussion |
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Several pathological conditions, such as diabetes mellitus25 and acute lung injury,26–28 are associated with enhanced microvascular 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.3–7 The pericyte, first described by Rouget3 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,14 gap junctions,29 and pericytic processes.6,29 Pericytes are contractile cells30 and this property has led to the suggestion that their effects on permeability are mechanical ones related to affecting the "open" state of endothelial intercellular junctions.9,11,16
The finding that the mesangial cell, the pericyte of the kidney, produces vascular permeability factor17 forced a reconsideration 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 organelle.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.31 We have proposed that the pericyte is the regulator of the movement of fluid across the microvasculature.18 As a first step in testing this hypothesis, we proposed that PMA, an agent that produces increased microvascular permeability in intact animal models18–21,32 enhances microvascular permeability via release of vascular permeability factor from microvascular pericytes.
In 1995, Aiello et al33 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 neovascularization associated with several disease states.33 Shortly thereafter, Takagi et al34 reported that the hypoxic induction of VEGF in retinal pericytes was mediated by adenosine. In the study presented here, we isolated, cultured, and purified bovine retinal pericytes. For purification, the use of 3G5 antibody-coated magnetic microspheres permitted the isolation of pericytes free of other microvascular cells. The pericytes were identified morphologically and by immunofluorescent staining for -smooth muscle actin and with 3G5 antibody. In initial experiments, L-NAME and Ang II, which had been reported to increase the expression of mRNA for VEGF in other systems, viz, lung35 and vascular smooth muscle,36 respectively, as well as PMA were used to stimulate pericytes. Only in the case of PMA was an increase in mRNA for VEGF identified. In subsequent experiments, mRNA for VEGF was found to be upregulated in a dose-dependent manner by PMA (10-9 to 10-6 mol/L). In the experiments reported here, we did not address the discrepancy between our results, which did not show upregulation of mRNA for VEGF in the pericytes in response to Ang II, and similar experiments in vascular smooth muscle, which revealed upregulation of VEGF in response to Ang II. Bovine retinal pericytes37 as well as human mesangial cells38 have been demonstrated to possess receptors for Ang II. In experiments in which pericytes were isolated and purified in a manner similar to the procedures we used, save for the purification step using 3G5 monoclonal antibody,23 Ang II was reported to attenuate pericyte relaxation in response to increasing the partial pressure of carbon dioxide in the solution bathing the pericytes.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 is required for Ang II to function as an agonist for upregulation of mRNA for VEGF in the pericyte, or that Ang II is not an agonist for upregulation of mRNA for VEGF in the pericyte.
The present study has not defined the mechanism whereby PMA regulated VEGF gene expression in pericytes. However, PMA is a protein kinase C agonist,40–42 which implicates a second messenger pathway involving protein kinase C. Indeed the results of both Aiello et al33 and Takagi et al34 are consistent with that interpretation, for both hypoxia43 and adenosine44 affect protein kinase C. The findings reported here are consistent with previous reports on signal transduction pathways of the VEGF gene.45,46
In conclusion, the results of our studies show that VEGF mRNA expression is upregulated by PMA stimulation in pericytes. This suggests that pericytes may participate in the increased microvascular permeability in conditions such as the adult respiratory distress syndrome or diabetes mellitus by increasing VEGF synthesis.
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Acknowledgments |
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This work was supported by an American Heart Association Grant (Missouri Affiliate) and by National Institutes of Health (National Heart, Lung and Blood Institute) Grants HL51298 and HL52675. We are indebted to Dr. Joseph J. Baldassare for guiding us in the molecular biological techniques used in this work. We thank W. Jo Schreiweiss for her excellent technical assistance.
Received September 18, 1997; first decision October 16, 1997; accepted October 29, 1997.
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