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(Hypertension. 1997;30:134-139.)
© 1997 American Heart Association, Inc.

Articles

Increased Transforming Growth Factor-ß Production and Gene Expression by Peripheral Blood Monocytes of Hypertensive Patients

Ettore Porreca; Concetta Di Febbo; Gabriella Mincione; Marcella Reale; Giovanna Baccante; Maria Domenica Guglielmi; Franco Cuccurullo; ; Giulia Colletta

From the Department of Internal Medicine (E.P., C. Di F., G.B., M.D.G., F.C.) and Institutes of Human Pathology and Social Medicine (G.M., G.C.) and Immunology (M.R.), University of Chieti (Italy), Medical School.

Correspondence to Dr Ettore Porreca, Laboratorio di Fisiopatologia Medica Centro Servizi Biomedici, Via dei Vestini, Università G. D'Annunzio, 66013 Chieti, Italy.

	Abstract

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Abstract Cultured human peripheral blood monocytes are known to secrete and express transforming growth factor-ß (TGF-ß), a multifunctional cytokine that can be involved in myocardial and vascular remodeling. In addition, monocytes/macrophages have been demonstrated to be colocalized with fibrosis of hypertrophied heart and in the vascular wall of hypertensive vessels. In this study, we tested TGF-ß production and mRNA expression in peripheral blood monocytes from hypertensive patients with myocardial hypertrophy and increased carotid myointimal thickness with respect to healthy normotensive control subjects. We found an increased TGF-ß activity in the conditioned medium of monocytes from hypertensive patients compared with control subjects as evaluated by inhibition of [³H]thymidine incorporation by mink lung epithelial cells (-83% and -18% in hypertensive and normotensive subjects; P<.001). Western blot analysis confirmed a significant difference in the amount of TGF-ß protein secreted in the conditioned medium of hypertensive patients compared with that of normotensive subjects. Finally, we also observed a 4.2- and 5.5-fold increase in the amount of TGF-ß₁ and TGF-ß₂ transcripts, respectively. Our results indicate an upregulation of the TGF-ß system in the peripheral blood monocytes of hypertensive patients with cardiovascular structural changes, suggesting a possible role of TGF-ß monocyte production in hypertensive disease.

Key Words: transforming growth factors • monocytes • heart hypertrophy • remodeling

	Introduction

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Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates many cellular functions, including cell growth and differentiation,^{1 2} modulation of extracellular matrix synthesis, and tissue repair,^{3 4 5} and may play an important role in regulating inflammation and immune responses.^{6 7} TGF-ß may also play a significant role in the vascular remodeling process⁸ and myocardial hypertrophy⁹ brought on by hypertension. TGF-ß₁ mRNA has been found to be increased in the aorta of deoxycorticosterone-salt hypertensive rats compared with normotensive rats,¹⁰ and vascular smooth muscle cells of spontaneously hypertensive rats show an altered responsiveness to TGF-ß₁ as well as autocrine TGF-ß expression.¹¹ In vivo data indicate that infusion of TGF-ß₁ or transfection of TGF-ß₁ cDNA¹² into injured arteries strongly accelerates lesion formation by increasing cellularity and extracellular matrix accumulation.

Myocardial fibrosis associated with hypertensive cardiac hypertrophy¹³ has been related to chronic elevations in circulating and/or local concentrations of stimulatory hormones.¹⁴ In this context, TGF-ß₁, fibronectin, and collagen mRNA are induced in the thoracic-banded hypertrophied rat myocardium,¹⁵ and TGF-ß₁ transcripts are increased in the myocyte fraction obtained from experimental myocardial hypertrophy induced by aortic constriction or after subcutaneous infusion of norepinephrine.¹⁶

In the present study, we evaluate the production and mRNA gene expression of TGF-ß in in vitro cultures of peripheral blood monocytes from hypertensive patients with cardiac hypertrophy and myointimal thickening with respect to healthy normotensive subjects. We used circulating leukocytic cells in accordance with the systemic nature of the structural remodeling process,¹⁴ since inflammatory cells are potential sources of TGF-ß in vivo,^{17 18 19} as well as monocytes that have been found in the subendothelium of spontaneously hypertensive rats,²⁰ in myocardial perivascular sites, and colocalized with myocardial fibrosis in hypertrophied hearts.^{21 22} Finally, hypertension may enhance the responsiveness of endothelium to factors that promote monocyte adhesion,²³ and atherosclerotic disease associated with hypertension is in turn characterized by an inflammofibroproliferative response in the vascular wall.²⁴ Our results show an increased capacity of monocytes of hypertensive patients to produce active TGF-ß. This increased secretion was due to an unstimulated increase in mRNA expression of TGF-ß₁ and TGF-ß₂. These findings suggest that monocyte synthesis of TGF-ß may play a role in cardiovascular remodeling of hypertensive disease.

	Methods

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Subjects
We studied 22 hypertensive patients (8 men, 14 women; mean age, 54±9 years) admitted to our Department of Internal Medicine with a history of previously untreated mild to moderate essential hypertension. All patients had appropriate clinical and laboratory evaluations to exclude secondary hypertension.²⁵ Noncardiovascular conditions associated with a possible pathogenetic role for TGF-ß (ie, malignancy, acute and chronic liver disease, and connective tissue disease) were excluded after complete medical and laboratory examinations. The control subjects consisted of 22 consecutive normotensive blood donors (8 men, 14 women; mean age, 51±8 years) without acute or chronic illness; none of them reported any symptoms related to the cardiovascular system.

The general biochemical evaluation included plasma glucose concentration; serum lipid profile; and measurement of serum potassium, serum and urinary creatinine, and inflammatory reactants (erythrocyte sedimentation rate, C-reactive protein, white blood cells). Biochemical parameters were measured by routine laboratory methods. The study was approved by the Ethics Review Committee of the University of Chieti; all participants gave written informed consent.

Echocardiography and Carotid Ultrasonography
All subjects underwent M-mode, two-dimensional, and Doppler ultrasound performed with an ultrasound imaging system (Hewlett-Packard 77030A) equipped with a 2.5-MHz transducer. Left ventricular (LV) dimensions were obtained from two-dimensionally guided M-mode tracings according to the recommendation of the American Society of Echocardiography.²⁶ LV mass was calculated according to Penn Convention recommendations.²⁷ LV mass was indexed by body surface area (LV mass index). The relative wall thickness was measured at end diastole as the ratio of 2xPosterior Wall Thickness/LV Internal Dimensions.

Arterial wall thickness was evaluated by carotid ultrasonography performed with a 7.5-MHz transducer.²⁸ Intima-media thickness was measured in both right and left common carotid arteries at least 1 cm below the carotid bifurcation. The intima-media thickness was defined as the distance from the leading edge of the lumen-intima interface of the far wall to the leading edge of the media-adventitia interface of the far wall. Measurements were taken at end diastole by electrocardiographic triggering. The mean of six measurements was used to derive an estimate of the overall intima-media thickness of the common carotid arteries. A repeat scan was made after 1 week to determine the reproducibility of the measurements. The coefficient of variation was 6.7%.

Isolation of Blood Monocytes and Cell Cultures
Peripheral heparinized blood was diluted 1:1 with normal saline, and mononuclear cells were separated by a Ficoll-Hypaque gradient (Pharmacia LKB Biotechnology Inc).^{29 30} Mononuclear cells were recovered at the interface, and peripheral blood mononuclear cells were rendered plasma-free and platelet-poor by washing three times with HEPES-buffered (10 mmol/L) Hanks' balanced salt solution and resuspended at the desired concentration in RPMI-1640 medium (GIBCO) supplemented with 0.3 mg/mL L-glutamine, 80 µg/mL gentamicin, and 5% heat-inactivated endotoxin-free fetal calf serum. Aliquots (2.5-mL) were seeded into Petri dishes and incubated at 37°C in a 5% CO₂–humidified atmosphere for 60 minutes. After that time, the supernatants, which contained nonadherent cells, were discarded, and the remaining adherent monocytes (95% purity determined by fluorescein isothiocyanate–labeled OKM5 staining) were cultured in RPMI-1640 medium supplemented with 0.3 mg/mL L-glutamine, 80 µg/mL gentamicin, and 1% endotoxin-free fetal calf serum at a density of 3x10⁶/mL in T75 flasks (Nunc). All cell cultures were maintained at 37°C in a controlled humidified 95% air/5% CO₂ atmosphere. Viability of the cultured cells was determined by trypan blue exclusion. The culture medium contained less than 10 pg/mL of lipopolysaccharides, as determined by the Limulus amebocyte lysate test.

Preparation of Monocyte Conditioned Medium and Mink Lung Epithelial Cell DNA Synthesis Bioassay for Active TGF-ß
Conditioned medium (CM) from equal cell numbers of cultured monocytes of patients and healthy control subjects was harvested after 24 hours and stored at -20°C before assay. As most TGF-ß is secreted in a latent form that is biologically inactive,³¹ the monocyte CM was activated by acidification before the assay using a standard protocol to examine the production of active TGF-ß. Briefly, CM was acidified with 10% (vol/vol) of 1 mol/L hydrochloric acid and incubated at room temperature for 60 minutes. An equal amount of 1 mol/L sodium hydroxide solution was added for neutralization, and the CM was added to the assay plates. TGF-ß activity by monocytes was examined by a bioassay sensitive to TGF-ß₁ -ß₂, and -ß₃.³² CCL-64 mink lung epithelial cells (American Type Culture Collection) were plated in Dulbecco's modified Eagle's medium plus 10% fetal calf serum at a density of 8x10³ cells per 0.2 mL in 96-well plastic plates. After 20 hours, the medium was replaced, and aliquots of the test samples were added in quadruplicate to monolayers. After 22 hours, DNA synthesis was determined by pulsing for 4 hours with 0.5 µCi of [³H]thymidine (2 µCi/mL, Amersham International plc). The amount of radioactivity in the cells exposed to the test samples was determined with a liquid scintillation counter (Packard Instruments). A standard curve was constructed with increasing concentrations of purified platelet human TGF-ß₁ (R&D System) in each experiment. In preliminary experiments, we tested TGF-ß activity in the acidified CM of monocyte cultures of control subjects and hypertensive patients at different dilutions. About 50% inhibition was observed at dilutions of 1:4 and 1:16 CM for control subjects and hypertensive patients, respectively. To test all samples, we used a dilution of 1:8, which accurately detected TGF-ß activity in the linear portion of its standard curve. On the basis of the standard curve, TGF-ß levels were calculated by multiplying the measured TGF-ß concentrations by the dilution factor and were expressed as nanograms per milliliter.

For determination of the specificity of the inhibitory response, a purified IgG fraction of rabbit polyclonal neutralizing anti–TGF-ß (Pan-specific TGF-ß neutralizing antibody, R&D System) was added to the CM 1 hour before its addition to the mink lung epithelial cells.

Western Blot Analysis
CM of cultured mononuclear cells was collected after 24 hours, centrifuged at 3500 rpm at 4°C, and concentrated with centrifuge concentrators (Centripor; molecular weight cutoff, 10 000 D; Spectrum Microfuge). Aliquots (20 µL) of concentrated and desalted supernatants were resuspended in 10 µL sample buffer (50 mmol/L Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 15% acrylamide gel for 60 minutes at 180V using a minigel vertical apparatus (Bio-Rad Laboratories, SrL). Some samples were reduced with sample buffer plus 5% ß-mercaptoethanol. For reference purposes, 10 ng human platelet purified TGF-ß₁ (R&D System) was coelectrophoresed in a separate lane. Size-separated proteins were blotted onto Hybond ECL membrane (Amersham International), saturated with a solution of 5% nonfat milk in phosphate-buffered saline/0.1% Tween-20 (PBS/Tween-20), and incubated for 60 minutes with rabbit polyclonal IgG antibody directed against human TGF-ß (1:2000) (R&D System). The filters were washed three times in PBS/Tween-20 and incubated for 60 minutes with rabbit horseradish peroxidase–labeled conjugated anti-rabbit IgG (1:3000) for 60 minutes at room temperature. The immune complex was visualized with the ECL Western blot detection system (Amersham International).

RNA Extraction and Northern Blot Analysis
RNA was purified from cultured adherent monocytes by a modification of the guanidine hydrochloride extraction method.³³ Total RNA (5 µg) was fractionated on a 1% formaldehyde agarose gel, transferred to nylon membranes (Hybond N, Amersham International), and hybridized following the standard procedure. Gel-purified fragments of DNA random primed with ³²P (2x10⁸ cpm/µg) were used for hybridization. The probe used was human TGF-ß cDNA, 1.3-kb fragment, which recognizes TGF-ß₁ and TGF-ß₂ isoforms (a kind gift of Dr M.L. McGeidy, Laboratory of Tumor Immunology and Biology, NIC, Bethesda, Md).

The sizes of the transcripts were indicated as relative to 18S and 28S rRNA, which were assumed to be 1.8 and 5.4 kb, respectively. Autoradiograms were prepared using exposure times of 18 hours. To compare the TGF-ß mRNA signals, we used a cDNA probe for GAPDH (American Type Culture Collection). A densitometer (Ultrascan XL, Pharmacia LKB) was used for normalization. The autoradiograms were scanned and peak areas measured for relative mRNA levels (TGF-ß mRNA/GAPDH) in the samples tested. Values were stored in a spreadsheet software program that produced a graphic representation of the signals.

Statistical Analysis
All data are reported as mean±SD. The significance of differences between the two groups was evaluated by the Mann-Whitney U test or Student's t test for unpaired data. One-way ANOVA and the Student-Newman-Keuls test were performed when appropriate. A value of P<.05 was considered statistically significant.

	Results

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Clinical, Biochemical, Echocardiographic, and Carotid Ultrasonographic Characteristics
The Table shows clinical, biochemical, and ultrasonographic findings in the control normotensive subjects and hypertensive patients. There were no significant differences between groups in sex, age, smoking habits, serum lipids values, platelets, and white blood cell numbers. Biochemical parameters of inflammatory reaction did not change in either group. By design, the hypertensive patients had significantly higher systolic (160±12 versus 124±10 mm Hg, P<.0001), diastolic (102±6 versus 72±9, P<.0001), and mean arterial (121±7 versus 89±8 mm Hg, P<.0001) blood pressures than their normotensive counterparts. LV mass index increase significantly in the hypertensive patients with respect to control subjects (130±36 versus 104±31 g/m², P<.05), as did relative wall thickness (48.7±7.6 versus 40±10, P<.005) and intima-media thickness (0.97±0.13 versus 0.69±0.11 mm, P<.0001).

View this table: [in this window] [in a new window]   Table 1. Baseline Clinical, Biochemical, and Ultrasonographic Parameters of Normotensive Subjects and Hypertensive Patients

Detection of Biological Activity and TGF-ß Protein in Medium From Monocyte Cultures
Mink Lung Epithelial Cell DNA Synthesis
It is well known that isolated monocytes secrete TGF-ß in a latent form that can be activated by acidification. According to the study by Assoian et al,¹⁸ the activity of untreated CM from our monocyte cultures was very low, as tested in the mink lung DNA synthesis assay. Thus, in all 44 subjects, we examined the production of active TGF-ß in the monocyte CM after acidification before the bioassay using a standard protocol, as described in "Methods." Fig 1 (top) shows that the mink lung cells responded to purified TGF-ß₁ with a dose-dependent decrease in DNA synthesis, expressed as a percentage of basal [³H]thymidine incorporation (5400 cpm). CM from monocyte cultures from hypertensive patients and control subjects was added to parallel cultures of mink lung cells, and the effects on DNA were synthesis examined. CM of monocyte cultures from hypertensive patients and control subjects inhibited mink lung epithelial cell DNA synthesis to 17±7% and 82±30% of control values, respectively (P<.001) (Fig 1, top, open circles). Using the standard curve generated with purified TGF-ß₁, these levels of inhibition equated to 1.76±0.2 ng/mL per 10⁶ cells of TGF-ß activity in the medium of normotensive subjects, which increased 2.7-fold in the medium of hypertensive patients (4.8±0.5 ng/mL per 10⁶ cells, P<.001).

View larger version (22K): [in this window] [in a new window]   Figure 1. Bioassay of transforming growth factor-ß (TGF-ß) activity in conditioned medium (CM) of monocyte cultures from hypertensive patients and normotensive subjects. CM from cultures of peripheral blood monocytes was collected after 24 hours and acidified for mink lung epithelial cell bioassay. Mink lung cells respond to purified human platelet TGF-ß1 with dose-dependent decreases of DNA synthesis (top, ) expressed as percentage of basal [3H]thymidine incorporation (5400 cpm). CM from monocyte cultures was added to parallel cultures of mink lung cells, and the effects on DNA synthesis were examined (top, ). Comparison with the standard curve and multiplication by the dilution factor (1:8) yielded TGF-ß concentration in CM of monocyte cultures. Open circles represent mean values obtained from multiple sample (n=22 patients for each group). For each sample, four measures of [3H]thymidine incorporation were made. Bottom, Effect of a pan-specific TGF-ß neutralizing antibody (Ab-anti TGFß) (0.1, 0.5, 5, and 10 µg/mL) on [3H]thymidine uptake by mink lung cells induced by CM (c.m. in the figure) of monocyte cultures from hypertensive patients. The antibody was added to CM 1 hour before its addition to mink lung epithelial cells. Results are mean±SD from four hypertensive patients. Data represent the average of quadruplicate determinations. Nonspecific rabbit IgG (10 µg/mL) was used for comparison. NS indicates nonsignificant versus control (C) by the Student-Newman-Keuls test.

The growth-inhibitory activity in the CM of monocytes was due to TGF-ß because pretreatment of CM with a neutralizing antibody abolished the growth-inhibitory effect. Fig 1, bottom, shows that the inhibitory effect on [³H]thymidine incorporation observed for a sample from a hypertensive patient was abolished after coincubation with 10 µg/mL of neutralizing anti–TGF-ß. The same neutralizing activity was observed for the small inhibition on [³H]thymidine incorporation observed in control CM (data not shown).

Western Blot Analysis
To verify whether the increase in TGF-ß activity was due to an increase of the protein growth factor secreted in the medium, we performed Western blot analysis in six subjects per group. CM secreted by human peripheral blood monocytes from control subjects and hypertensive patients was collected after 24 hours of culture, concentrated, and subjected to Western blot analysis. With the use of a rabbit antibody prepared against several isoforms of TGF-ß, a 25-kD peptide was detected in the medium of both normotensive subjects and hypertensive patients (Fig 2). According to the disulfide-linked 12.5-kD peptide structure,² reduction with ß-mercaptoethanol resulted in a shift to a 12.5-kD form in the monocyte TGF-ß. An increase in the amount of immunoreactive peptide was seen in the medium of monocyte cultures from hypertensive patients compared to that from normotensive subjects. Densitometric analysis of the Western blot showed a fourfold increase in the amount of TGF-ß secreted by cultured monocytes from hypertensive patients with respect to control subjects (P<.001).

View larger version (38K): [in this window] [in a new window]   Figure 2. Representative Western blot analysis of transforming growth factor-ß (TGF-ß) activity in conditioned medium from human peripheral blood monocytes of representative normotensive control subject and hypertensive patient. Aliquots of conditioned medium were concentrated and prepared for electrophoresis on a 15% polyacrylamide gel. Samples were run in the presence or absence of ß-mercaptoethanol as indicated and incubated with rabbit IgG antibody against TGF-ß. Bound antibody was detected with a horseradish peroxidase–labeled conjugated anti-rabbit IgG. Purified human platelet TGF-ß1 (10 ng) was electrophoresed for comparison.

TGF-ß mRNA Expression
To study whether the increase in TGF-ß production in hypertensive patients was due to a transcriptional upregulation, we carried out a Northern blot experiment using a cDNA probe that recognizes both TGF-ß₁ and TGF-ß₂ mRNA sequences. Since previous studies showed similar levels of TGF-ß₁ transcripts in both activated and nonactivated monocytes,^{18 19} we tested "constitutive" mRNA expression in our peripheral blood monocytes. Fig 3 shows representative Northern blot analysis of TGF-ß mRNA of three hypertensive patients and three control subjects after 24 hours of incubation. TGF-ß mRNA levels were calculated in relation to GAPDH levels in the same sample. A 2.4- and 6.5-kb mRNA for TGF-ß₁ and TGF-ß₂ were present in control subjects and hypertensive patients. A significant increased amount of TGF-ß₁ and TGF-ß₂ transcript was detected in the monocytes of hypertensive patients compared with those of control subjects. TGF-ß₁ and TGF-ß₂ mRNA contents in 15 patients and 15 normotensive control subjects were normalized to that of GAPDH mRNA as described in "Methods" and expressed as mean densitometric absorbance units (Fig 4). The values of the densitometric ratios (TGF-ß₁ and TGF-ß₂/GAPDH mRNA) showed mean increases of 4.2- and 5.5-fold, respectively, in cultured monocytes of hypertensive patients compared with control subjects.

View larger version (33K): [in this window] [in a new window]   Figure 3. Representative Northern blot analysis of transforming growth factor-ß1 (TGF-ß1), TGF-ß2, and GAPHD mRNAs in human peripheral blood monocytes of three hypertensive patients and three normotensive control subjects. Mononuclear cells were cultured for 24 hours in serum-free conditions. Total RNA (5 µg per lane) was electrophoresed on 1.5% formaldehyde agarose gels, transferred to nitrocellulose, and hybridized with a 32P random prime–labeled TGF-ß cDNA. Sizes of the transcripts were determined by comparison with 28S and 18S rRNA (not shown). Molecular size markers are shown on the right.

View larger version (11K): [in this window] [in a new window]   Figure 4. Monocyte transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 mRNA densitometric expression in 15 normotensive control subjects (open bars) and 15 hypertensive patients (solid bars). For each patient, TGF-ß1 and TGF-ß2 mRNA contents were normalized to that of GAPHD mRNA. Mean±SD are shown of densitometric units of TGF-ß1 and TGF-ß2/GAPDH blots. *P<.0001.

	Discussion

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We designed the current study to examine TGF-ß production and its mRNA expression by human monocytes of hypertensive patients with cardiovascular structural changes. We tested a basic aspect of monocyte behavior in ex vivo cultures of peripheral blood monocytes of hypertensive patients with concentric hypertrophy and myointimal thickening with respect to healthy subjects. In CM from nonstimulated monocytes (24-hour collection), we first evaluated the biological activity of TGF-ß by bioassay on mink lung epithelial cells. A significantly greater inhibitory effect in [³H]thymidine incorporation of mink lung cells was observed in the CM of monocytes from hypertensive patients than in those from control subjects. To verify whether the increase in TGF-ß activity was due to transcriptional regulation, we performed Northern blot analysis for TGF-ß mRNA expression.

We found a significant mean increase of TGF-ß₁ (4.2-fold) and TGF-ß₂ (5.5-fold) transcripts in monocyte cultures of hypertensive patients with respect to normotensive control cultures after 24 hours of incubation. Western blot analysis performed on the CM from monocyte cultures further confirmed the previously observed differences in the mRNA amount and biological activity, showing a significant increase in the corresponding protein in cultures of monocytes from hypertensive patients. Thus, a spontaneous increase in TGF-ß production by mononuclear cells of hypertensive patients correlated with and enhanced expression of TGF-ß mRNA.

Our results support the hypothesis that monocytes/macrophages possibly recruited into the cardiovascular tissue may represent a significant source of TGF-ß. Thus, even though TGF-ß induction in hypertrophied myocardium after pressure overload has been demonstrated to be associated with the myocyte fraction,¹⁹ colocalization of fibroblastic activity with lymphocytes and macrophage infiltration in the hypertrophied myocardium suggest the possibility of a leukocytic origin of myocardial TGF-ß, which may play a role in the process of myocardial fibrosis.

Furthermore, upregulation of the TGF-ß system in monocytes of hypertensive patients supports the view of cellular (monocyte) involvement in the pathogenesis of atherosclerotic lesions associated with hypertension.^{34 35} In this setting, the observed association between LV hypertrophy and carotid atherosclerosis has been demonstrated to be independent of hypertensive blood pressure values,³⁵ and attempts to reduce cardiovascular complications due to atherosclerosis with antihypertensive treatment have not always been successful.³⁴ The significant increase in monocyte TGF-ß₂ mRNA observed in our study agrees with other studies showing the involvement of TGF-ß₂ in vascular remodeling and in vivo fibrosis^{36 37 38} and further supports a role for the TGF-ß system in the pathophysiology of vascular matrix remodeling.⁸ We did not evaluate the mechanisms involved in the increased expression of TGF-ß mRNA levels in the monocytes of hypertensive patients. However, induction of TGF-ß mRNA by autocrine or paracrine TGF-ß has been demonstrated in fibroblasts and vascular smooth muscle cells³⁹ ; also, the growth-promoting effect of angiotensin has been shown to occur when angiotensin activates autocrine and paracrine growth factors, including TGF-ß.^{40 41} In this context, in the present study, we did not test the relationship between TGF-ß production/expression and the cardiovascular renin-angiotensin system, which also plays a significant role in cardiac and vascular hypertrophy and remodeling.⁴² However, interestingly, converting enzyme inhibitors prevent the increased monocyte/macrophage traffic throughout the arterial wall as well as myocardial fibrosis²³ ; angiotensin II type 1 receptor antagonists inhibit the gene expression of TGF-ß₁ and extracellular matrix in cardiac and vascular tissue of hypertensive rats⁴³ ; and angiotensin II receptors have been demonstrated in human monocytes.⁴⁴ Thus, a possible relationship between the renin-angiotensin and TGF-ß systems, possibly at the monocyte level, cannot be excluded.

In conclusion, our findings indicate that the source of TGF-ß in cardiovascular tissues during hypertensive disease may be of macrophage/monocytic origin. The increased TGF-ß production associated with increased mRNA expression observed in hypertensive patients demonstrates an upregulation of the TGF-ß system and also supports the possibilities that macrophage/monocytic cells infiltrate hypertrophic myocardium or that the vascular wall plays a significant role in the cardiovascular remodeling process.

	Acknowledgments

 
This work was partially supported by the ACRO Project of the National Research Council (CNR) and by AIRC. G.M. is supported by a fellowship from AIRC.

Received October 7, 1996; first decision October 29, 1996; accepted November 29, 1996.

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