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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2000 Mar 21;97(7):3479–3484. doi: 10.1073/pnas.050420897

Transforming growth factor-β1 hyperexpression in African-American hypertensives: A novel mediator of hypertension and/or target organ damage

Manikkam Suthanthiran *, Baogui Li *, Jong O Song *, Ruchuang Ding *, Vijay K Sharma *, Joseph E Schwartz †, Phyllis August *,‡
PMCID: PMC16265  PMID: 10725360

Abstract

Hypertension, a remediable risk factor for stroke, cardiovascular disease, and renal failure, affects 50 million individuals in the United States alone. African Americans (blacks) have a higher incidence and prevalence of hypertension and hypertension-associated target organ damage compared with Caucasian Americans (whites). Herein, we explored the hypotheses that transforming growth factor-β1 (TGF-β1) is hyperexpressed in hypertensives compared with normotensives and that TGF-β1 overexpression is more frequent in blacks compared with whites. These hypotheses were stimulated by our recent demonstration that TGF-β1 is hyperexpressed in blacks with end-stage renal disease compared with white end-stage renal disease patients and by the biological attributes of TGF-β1, which include induction of endothelin-1 expression, stimulation of renin release, and promotion of vascular and renal disease when TGF-β1 is produced in excess. TGF-β1 profiles were determined in black and white hypertensive subjects and normotensive controls and included circulating protein concentrations, mRNA steady-state levels, and codon 10 genotype. Our investigation demonstrated that TGF-β1 protein levels are highest in black hypertensives, and TGF-β1 protein as well as TGF-β1 mRNA levels are higher in hypertensives compared with normotensives. The proline allele at codon 10 (Pro10) was more frequent in blacks compared with whites, and its presence was associated with higher levels of TGF-β1 mRNA and protein. Our findings support the idea that TGF-β1 hyperexpression is a risk factor for hypertension and hypertensive complications and provides a mechanism for the excess burden of hypertension in blacks.

Hypertension, a prevalent and remediable risk factor for stroke, cardiovascular disease, and renal failure, affects 50 million individuals in the United States alone (1). African Americans (blacks) have a higher incidence and prevalence of hypertension compared with Caucasians (whites): 33% of blacks are affected, compared with 25% of whites. Even more striking is the disparity in the prevalence of severe or malignant hypertension; it is estimated to be five to seven times more prevalent in blacks compared with whites (2). Morbidity attributable to hypertension is also more prevalent in blacks. For example, left ventricular hypertrophy was three times more common in blacks compared with whites in the Hypertension Detection and Follow-Up Program (HDFP) (3). After accounting for established baseline risk factors, blacks still have a 38% greater risk of incident ischemic stroke compared with whites, according to the Atherosclerosis Risk in Communities study (4). End-stage renal disease (ESRD), a devastating medical illness of extraordinary personal suffering and societal costs, is four times more frequent in blacks, and ESRD attributable to hypertension is 20 times more frequent in blacks (5). Data from the Multiple Risk Factor Intervention Trial demonstrated that for every level of blood pressure control, blacks are at increased risk for the development of renal disease (6). Finally, and perhaps most disturbing, are the observations that aggressive blood pressure control has not uniformly led to similar renal-protective benefits in blacks and whites (7, 8).

Genetically determined renal and hormonal characteristics as well as several environmental influences have been proposed as contributing to the excess burden of hypertension in blacks. To date there is no unifying hypothesis for the overabundance of hypertensive diseases in the black population.

Transforming growth factor-β1 (TGF-β1) is a 25-kDa homodimeric protein secreted by several cell types, including peripheral blood mononuclear cells (PBMC), endothelial cells, vascular smooth muscle cells, platelets, and also renal cells (9, 10). It facilitates extracellular matrix assembly by stimulation of its synthesis and prevention of matrix degradation and is important in tissue repair and remodeling. Overproduction, on the other hand, is an important mechanism for fibrogenesis (9, 10). TGF-β1 has been implicated in several of the long-term chronic sequelae of poorly controlled hypertension, including left ventricular hypertrophy (11), hypertensive vascular remodeling (12), and ESRD (13). Angiotensin II is a known stimulus of TGF-β1 production; thus, TGF-β1 may be an important mediator of angiotensin II-induced hypertensive damage (14). Although much of TGF-β1's biological action is mediated in an autocrine or paracrine fashion, data from TGF-β1 transgenic mice have demonstrated that high-circulating levels can mediate renal fibrosis and progressive loss of function (15).

Recent data also suggest that TGF-β1 may have a direct pathogenic role in elevated blood pressure. TGF-β1 stimulates the expression of mRNA encoding endothelin-1 in vascular endothelial cells (16) and also increases renin release from juxtaglomerular cells in the kidney (17).

We have demonstrated that circulating levels of TGF-β1 are higher in blacks with ESRD compared with white ESRD patients and that TGF-β1 levels are also correlates of blood pressure in patients with ESRD (18, 19). We hypothesized that the increased frequency of hyperexpression of TGF-β1 may contribute to the excess burden of ESRD in blacks. We also have suggested that hyperexpression of TGF-β1 might be genetically determined, because DNA polymorphisms in codon 25 of the TGF-β1 gene have been associated with hypertension (20) and codon 10 polymorphisms have been associated with lung fibrosis (21).

In this study, we explored the hypotheses that, independent of renal disease, TGF-β1 is hyperexpressed in hypertensives compared with normotensives and that TGF-β1 hyperexpression is more frequent in blacks compared with whites. Our postulate was tested by evaluating TGF-β1 profiles in hypertensive and normotensive subjects. TGF-β1 profiling included determination of circulating levels of TGF-β1 protein, quantification of steady-state levels of TGF-β1 mRNA in PBMC, and analysis of TGF-β1 DNA codon 10 polymorphisms in hypertensive and normotensive subjects.

Methods

Subjects.

Hypertensive subjects were recruited from the outpatient clinic at the Hypertension Division of Weill Medical College of Cornell University. Subjects were included if blood pressure (average of three determinations) was greater than 140/90 mmHg (1 mmHg = 133 Pa) on at least two occasions if patients were off medications. For patients taking antihypertensive medication, records were reviewed to ascertain the validity of the diagnosis of hypertension, and patients were included if blood pressure had been greater than 140/90 mmHg on two occasions in the past, before treatment. Patients with renal insufficiency and/or diabetes (serum creatinine ≥2 mg/dl) were excluded. Normal subjects were recruited from hospital employees and were required to have a blood pressure of <140/90 mmHg on three determinations. Blood pressure was measured in the seated position according to American Heart Association guidelines (22). Individuals with significant comorbidity (cancer, autoimmune disease, or active infection) were excluded. The protocol was approved by the Committee for Human Rights in Research at Weill Medical College of Cornell University. Oral consent was obtained for collection of venous blood.

Evaluation of TGF-β1 Profile.

TGF-β1 profiling consisted of measurement of serum TGF-β1 protein levels, quantification of mRNA in PBMC, and genotyping of codon 10. Peripheral venous blood was obtained and sera were isolated and stored at −70°C until assayed for TGF-β1 protein. PBM were isolated by Ficoll/Hypaque centrifugation; DNA was extracted from leukocytes for TGF-β1-genotyping analysis.

TGF-β1 codon 10 genotyping by amplification-refractory mutation system–PCR (ARMS-PCR).

ARMS-PCR is an allele-specific PCR that permits the ready detection of single-nucleotide polymorphisms (23, 24). Our design of ARMS primers for the identification of codon 10 genotypes is based on the principle that oligonucleotides that are complementary to a target DNA sequence except for a mismatched 3′ terminus will not function as PCR primers (Fig. 1). Our primers for codon 10 genotyping also were designed to include a penultimate mismatch. This inclusion enhances further the stringency of ARMS-PCR. Two complementary reactions were established for each allele and consisted of target DNA, allele-specific ARMS primer, and the common primer. PCR products were resolved by agarose gel electrophoresis. A thermal cycler was used, and each experiment had negative and positive controls for the target allele.

Figure 1.

Figure 1

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Design, location, and sequences of the oligonucleotide primers for the detection of codon 10 polymorphisms by ARMS-PCR. A penultimate mismatch was introduced into the sense (common) primer as well as into the allele-specific antisense ARMS primers.

Quantification of TGF-β1 mRNA.

A 347-bp TGF-β1 competitor DNA template was constructed for quantification of TGF-β1 mRNA by competitive quantitative PCR. In brief, an oligonucleotide primer pair was designed to amplify a region in the TGF-β1 gene that contains a Sau3A1 restriction site. The Sau3A1 digestion of the 255-bp TGF-β1 PCR product yielded 168- and 87-bp subfragments that were annealed with a 92-bp DNA insert synthesized in vitro to have cohesive ends for the Sau3A1 restriction site at 5′ and 3′ ends. The phosphorylated 92-bp DNA fragment was ligated with the 168- and 87-bp DNA fragments by using Escherichia coli DNA ligase. After ligation, the mixture was run on a 2% low-melting-point agarose gel, and the 347-bp fragment was eluted and purified.

The cDNA for quantification by competitive PCR was synthesized in a reverse transcription reaction mixture containing 1 μg of total RNA (isolated with the Qiagen RNEasy Kit; Qiagen, Chatsworth, CA), 100 ng of random hexanucleotide primers, and 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL). A constant amount of the cDNA then was coamplified with known concentrations of the competitor DNA construct for 32–36 cycles by using TGF-β1 gene-specific primer pairs (0.2 μM each) in a reaction mixture containing 1× Taq buffer, 1 unit of Taq DNA polymerase, and 40 μM of each dNTP. The PCR products were resolved by 2% agarose gel electrophoresis, visualized by ethidium bromide staining, and photographed. The negative of the photographs was analyzed by laser densitometry, and the absolute absorbance values of the PCR products were determined. The ratios of the absorbance of the relevant PCR product pairs (347-bp TGF-β1 competitor and 255-bp TGF-β1) were plotted against the concentrations of the competitor DNA used. The concentrations of mRNA thus were determined and expressed as attograms of TGF-β1 mRNA per μg of total RNA. mRNA encoding a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was quantified by using GAPDH-specific competitor template (25).

Measurement of TGF-β1 protein levels.

The biologically active TGF-β1 protein concentration was determined by using a solid-phase TGF-β1-specific sandwich ELISA (Promega) as described (26). The sera were activated by acidification and tested at 1:200 dilution. A TGF-β1 standard curve was constructed by using 1,000, 500, 250, 62.5, 31.25, and 15.6 pg/ml recombinant TGF-β1 protein, and a curve-fitting software program was used to quantify TGF-β1 protein concentration in the sera. The minimum level of detection of TGF-β1 with the sandwich ELISA was 25 pg/ml.

Statistical Analysis.

sas (statistical analysis software 6.1) was used for data analysis (27). One-way ANOVA was used for the comparison of multiple groups, and corrected (Bonferroni) P values were calculated to correct for type I experiment-wise error rate. A multiple regression analysis was used to estimate the independent effect of race on TGF-β1 protein levels controlling for gender and age. A multiple logistic regression analysis was used to estimate the effect of TGF-β1 protein levels on hypertension status controlling for race, gender, and age. The Mann–Whitney two-sample test was used to compare continuous variables, and categorical variables were analyzed by χ2 analysis.

Results

TGF-β1 Protein Concentration.

Quantification of TGF-β1 levels in sera obtained from hypertensive subjects and normotensive controls with a TGF-β1-specific ELISA demonstrated that TGF-β1 levels are higher in hypertensives compared with normotensives. The mean ± SEM TGF-β1 protein concentration was 261 ± 9 ng/ml in the hypertensive subjects (n = 61) and was 188 ± 7 ng/ml in the normotensive controls (n = 90) (P < 0.0001, Mann–Whitney two-sample test, Fig. 2A).

Figure 2.

Figure 2

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Serum TGF-β1 protein levels. Circulating levels of TGF-β1 protein were quantified by using a TGF-β1-specific sandwich ELISA. TGF-β1 protein levels, distinguished by diagnosis (hypertensive vs. normotensive) are shown in A. The mean ± SEM TGF-β1 level was 188 ± 7 ng/ml in normotensives (N) and was 261 ± 9 ng/ml in hypertensives (HT). TGF-β1 protein levels, distinguished by diagnosis as well as by race, are illustrated in B. The mean ± SEM TGF-β1 concentrations were 165 ± 6 ng/ml, 221 ± 12 ng/ml, 235 ng/ml, and 322 ng/ml in normotensive whites (NW), normotensive blacks (NB), hypertensive whites (HTW), and hypertensive blacks (HTB), respectively (P < 0.0001, ANOVA). The frequency distribution of TGF-β1 levels, distinguished by race and diagnosis, is shown in C.

Fig. 2B compares TGF-β1 protein levels across diagnosis (hypertensive or normotensive) and race (black or white). TGF-β1 levels were the highest in black hypertensives, and comparison of the mean TGF-β1 levels across the four groups demonstrated that the null hypothesis of equal group means should be rejected (P < 0.0001, ANOVA, Fig. 2B).

Bonferroni P values were calculated to correct for type 1 experiment-wise error rate in view of comparisons across multiple groups. The multiple comparisons showed that the TGF-β1 protein levels in black hypertensives (322 ± 16 ng/ml, n = 18) were higher than that in white hypertensives (235 ± 9 ng/ml, n = 43, P < 0.001), black normals (221 ± 12 ng/ml, n = 39, P < 0.001), and white normals (165 ± 6 ng/ml, n = 51, P < 0.001). The analysis also showed that TGF-β1 protein levels in black normals were significantly higher than those in white normals (P < 0.001), but not different from those in white hypertensives (P > 0.05).

The frequency distribution of TGF-β1 levels in the black and white normotensive and black and white hypertensive subjects is shown in Fig. 2C. Whereas 66.6% of the black hypertensives had TGF-β1 levels greater than 300 ng/ml, only 16.2% of white hypertensives had levels greater than 300 ng/ml (P < 0.00009, Fisher's exact test).

A multiple regression analysis was used to estimate the independent effects of race (black vs. white) on TGF-β1 protein levels, controlling for gender and age. Table 1 shows that race is a highly significant predictor of TGF-β1 levels. In this analysis, neither gender nor age was a significant determinant of TGF-β1 protein levels.

Table 1.

Race predicts TGF-β1 levels

Parameter Estimate SE T P
Intercept 205.86 27.80 7.40 0.0001
Blacks vs. whites 80.46 14.59 5.52 0.0001
Female vs. male 0.84 14.23 0.06 0.95
Age 0.31 0.47 0.65 0.52
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R2 = 0.260. 

A multiple logistic regression analysis then was used to estimate the effect of TGF-β1 protein levels on hypertension status controlling for race, gender, and age. This analysis demonstrated that TGF-β1 protein concentration is a significant predictor of diagnosis (P = 0.004). The estimated odds of an individual at the 75th percentile of TGF-β1 distribution (285 ng/ml) being hypertensive was 234% greater than that of an individual at the 25th percentile (187 ng/ml).

TGF-β1 mRNA Steady-State Levels.

Quantification of TGF-β1 mRNA levels in PBMC obtained from hypertensive subjects and normotensive controls showed that TGF-β1 mRNA was expressed at a significantly higher level in PBMC of hypertensive subjects (n = 55) compared with PBMC of normotensive controls (n = 41) (Fig. 3, P < 0.04, Mann–Whitney two-sample test). In a fashion similar to TGF-β1 protein levels, black hypertensives had the highest level of TGF-β1 mRNA. However, there was no significant difference in the TGF-β1 mRNA steady-state levels between black hypertensives and white hypertensives.

Figure 3.

Figure 3

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TGF-β1 mRNA steady-state levels in hypertensives and normotensives. The mean ± SEM TGF-β1 mRNA/GAPDH mRNA ratios, calculated after quantification of mRNA levels in PBMC, were 3.35 ± 0.81 and 4.51 ± 0.69 in normotensives (N) and hypertensives (HT), respectively (P < 0.04, Mann–Whitney two-sample test).

TGF-β1 Codon 10 Genotypes.

A total of 124 individuals were genotyped for TGF-β1 codon 10 by using the ARMS-PCR primers illustrated in Fig. 1. TGF-β1 codon 10 genotype of blacks differed significantly from that of whites in that there was an excess of proline allele (C = proline; T = leucine) at codon 10 (Pro10) in blacks compared with whites. The race-based difference in the relative frequencies of T/T, T/C, and C/C genotypes was significant (Table 2, likelihood ratio χ2 analysis, 8.67, P = 0.017). In blacks as well as whites, no significant deviation in codon 10 frequencies from Hardy–Weinberg expectation was observed.

Table 2.

TGF-β1 codon 10 genotypes of blacks and whites

Race/diagnosis (n) T/T T/C C/C
Blacks/normal (23) 13.04%  (3) 56.52%  (13) 30.43%  (7)
Whites/normal (35) 34.29%  (12) 48.57%  (17) 17.14%  (6)
Blacks/HT (21) 19.05%  (4) 52.38%  (11) 28.57%  (6)
Whites/HT (45) 37.78%  (17) 51.11%  (23) 11.11%  (5)
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TGF-β1 codon 10 genotypes were determined by using ARMS-PCR. The race-based difference in the relative frequency of T/T, T/C, and C/C genotypes was significant at P = 0.01. 

Correlations Between TGF-β1 Genotype and Expression.

TGF-β1 mRNA levels were higher in those genotyped as C/C or T/C at codon 10 compared with those genotyped as T/T. In 87 individuals (34 white hypertensives, 16 black hypertensives, 26 white normotensives, and 11 black normotensives) in whom codon 10 genotype and TGF-β1 mRNA levels were determined, the mean ± SEM TGF-β1 mRNA level in individuals genotyped as C/C or T/C was 5.06 ± 0.30 and was 3.53 ± 0.46 in those genotyped as T/T at codon 10 (Table 3, P < 0.0074, ANOVA).

Table 3.

Correlation between codon 10 genotypes and TGF-β1 mRNA levels

Codon 10 genotype TGF-β1 mRNA/GAPDH mRNA, mean ± SEM P
C/C + T/C 5.06  ±  0.30
T/T 3.53  ±  0.46 0.0074
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TGF-β1 mRNA levels and GAPDH mRNA levels were quantified by using gene-specific competitor constructs in competitive PCR and correlated with codon 10 genotype. mRNA steady-state levels (log-transformed) are from 87 individuals (34 white hypertensives, 16 black hypertensives, 26 white normotensives, and 11 black normotensives), who also were genotypes for codon 10. P value for the F statistic of a one-way ANOVA is shown. 

TGF-β1 protein concentrations were also higher in those genotyped as C/C or T/C at codon 10 compared with those genotyped as T/T. TGF-β1 protein levels were 16.25 ng/ml higher in those with the proline allele at codon 10 compared with those homozygous for the leucine allele at codon 10.

Discussion

The findings from this investigation are that TGF-β1 is overexpressed in hypertensive subjects compared with normotensive controls and that hyperexpression is more frequent in blacks compared with whites. Whereas a cause-and-effect relationship cannot be established from a cross-sectional study, our data regarding TGF-β1 at the levels of DNA, mRNA, and protein suggest a candidate mechanism for hypertension in humans and for the excess burden of hypertension and hypertensive complications in blacks.

Increased Frequency of Pro10 Allele in Blacks.

To date, seven single-nucleotide polymorphisms in the human TGF-β1 gene have been identified (20). These include three single-base substitutions in the upstream region of the TGF-β1 gene at positions −988 (C → A), −800 (G → A), and −509 (C → T); one in a nontranslated region at position +72 (C insertion); two single-base substitutions in the signal sequence at positions +869 (codon 10, T → C, Leu → Pro) and +915 (codon 25, G → C, Arg → Pro); and one in codon 263 (C → T, Thr → Ile). Strong linkage disequilibrium among six of the seven polymorphisms has been reported. Polymorphisms located in the signal peptide region regulate translation and/or vectorial transport of proteins (28). In view of our findings that TGF-β1 protein levels distinguished blacks from whites, we explored the hypothesis that codon 10 alleles, located in the signal peptide region of the TGF-β1 gene, would distinguish blacks from whites. Our focus on the TGF-β1 codon 10 polymorphism also was informed by the observation that codon 10 polymorphisms are correlates of pulmonary fibrosis, a potential consequence of TGF-β1 overexpression (21). That codon 10 genotyping would be informative also was suggested by an earlier observation that codon 25 alleles (also located in the signal peptide region) are correlates of blood pressure (20) and by our earlier demonstration that the G/G genotype at codon 25 is numerically higher in hypertensives compared with normotensives (19). Also, the reported allele frequencies at codon 10 permit appreciation of frequency differences between study populations by using a modest sample size.

The frequencies of T/T, T/C, and C/C genotypes found in our white subjects were similar to the frequencies observed in an earlier study of 1,192 white Europeans (20). The frequencies in our black subjects were different from that in the earlier study. We are unaware of studies of TGF-β1 DNA polymorphisms in black subjects and, thus, were unable to compare our data with those of others. The number of blacks genotyped in our study for codon 10, however, is small (n = 44). A larger study is required to better establish the race-dependent differences in TGF-β1 allele frequencies.

Our investigation also identified higher TGF-β1 mRNA steady-state levels in those with a Pro10 allele. Whereas a direct cause-and-effect relationship between codon 10 alleles and steady-state levels of TGF-β1 mRNA remains unproved at this time, it can be hypothesized that codon 10 is important in the regulated expression of the TGF-β1 gene, because polymorphisms at this location (signal sequence region) can influence protein secretion and TGF-β1 can regulate its own transcription via AP-1 sites located in its promoter (29). This idea also is supported by our finding that TGF-β1 protein concentrations are higher in those with the Pro10 allele compared with those homozygous for the Leu10 allele.

TGF-β1 mRNA Hyperexpression in Hypertensives.

A recent study of 22 hypertensive subjects with myocardial hypertrophy found that monocytes obtained from these patients expressed higher levels of mRNA encoding TGF-β1 compared with normal controls (30). Our observation that TGF-β1 mRNA steady-state levels are higher in hypertensive subjects without overt target organ damage compared with normotensive controls extends this earlier observation and suggests that TGF-β1 hyperexpression precedes the development of hypertensive complications.

TGF-β1 mRNA steady-state levels distinguished hypertensives from normotensives but not blacks from whites, unlike protein levels, which distinguished not only hypertensives from normotensives, but also blacks from whites. One potential explanation for the discordance might be the smaller sample size of the mRNA studies compared with protein determinations. An additional explanation, and one that we favor, is that multiple cell types, in addition to PBMC, contribute to circulating levels of TGF-β1 protein levels and prevent a strict correlation between PBM mRNA levels and TGF-β1 protein concentrations. Indeed, in our earlier study of cyclosporine-induced TGF-β1 hyperexpression we found a similar lack of correlation between circulating levels of TGF-β1 protein and PBMC TGF-β1 mRNA (31).

Race Is an Independent Predictor of TGF-β1 Protein Level, and TGF-β1 Levels Correlate with Hypertension Status.

In our earlier investigation, we found that TGF-β1 protein levels are higher in black patients with ESRD (18) compared with white patients with ESRD. In that study confined exclusively to ESRD patients, TGF-β1 levels also correlated with blood pressure. The current investigation extends these observations in a number of important ways and demonstrates, without the confounding variable of ESRD, that race is an independent predictor of TGF-β1 levels. In the current study, TGF-β1 levels were higher in blacks compared with whites, and the odds of being hypertensive were significantly higher in those with TGF-β1 levels in the 75th percentile compared with those in the 25th percentile. In view of the biological attributes of TGF-β1, a novel mechanism for the higher incidence and prevalence of hypertension and hypertension-associated target damage in blacks, is advanced by our finding that race predicts TGF-β1 protein levels and TGF-β1 levels are correlates of hypertension status.

How Might TGF-β1 Contribute to the Pathogenesis of Hypertension and Cardiovascular and Renal Complications?

We propose that TGF-β1 may contribute to the pathogenesis of hypertension by directly causing elevated blood pressure and/or by causing fibrosis and vascular injury. That TGF-β1 can raise blood pressure is supported by the observation that TGF-β1 stimulates the expression of endothelin-1, a potent vasoconstrictor (16). In this regard, it is worth noting that plasma endothelin-1 levels are higher in black hypertensives compared with white hypertensives (32). TGF-β1 also has been reported to stimulate renin release from juxtaglomerular cells, which may contribute to increased angiotensin II generation and blood pressure (17). An autoamplification loop that involves TGF-β1, endothelin-1, and the pressor peptide angiotensin II also can be envisioned in view of the observation that angiotensin II stimulates TGF-β1 expression. The observation that angiotensinogen levels are higher in blacks compared with whites and that the angiotensinogen promoter polymorphism (G/G at position −6), which correlates with a lower transcriptional rate, is more frequent in whites compared with blacks also may be relevant with respect to the interactions between the renin angiotensin system and TGF-β1. For example, increased angiotensinogen levels may generate excess angiotensin II, which then would lead to increased TGF-β1.

Hypertensive vascular pathology is a multicellular process, and fibrocellular hyperplasia and vascular smooth muscle proliferation and migration are some of the cardinal histologic features. In this context, it is worth noting that direct transfer of TGF-β1 gene into arteries results in fibrocellular hyperplasia (33) and that this multifunctional cytokine also can induce vascular smooth muscle cell hypertrophy (34). Transdifferentiation of vascular smooth muscle cells into chondrocytes and intimal growth also have been observed after transfection of the TGF-β1 gene into murine arteries (35). Differential sensitivity of vascular cells to TGF-β1 signaling also might be relevant because TGF-β1 is reported to potentiate platelet-derived growth factor (PDGF)-induced proliferation of vascular smooth muscle cells of the spontaneously hypertensive rat but not the vascular smooth muscle cells of the normotensive Wistar–Kyoto rat (36).

The biologic consequences of TGF-β1 are most likely realized by its acting in unison with other growth factors such as PDGF and fibroblast growth factor (9). In this context, it is worth noting that endothelin-1 and angiotensin II, each of which is regulated by TGF-β1, function not only as vasoconstrictors but also as growth factors for vascular cells (37–39).

Angiotensin II as well as other pressor agents also might regulate TGF-β1 expression via physical forces because shear stress can heighten TGF-β1 expression (40). TGF-β1 overexpression has been observed in the aorta of deoxycorticosterone-salt-sensitive, hypertensive rats (12, 41), and cardiac myocytes, subjected to the hypertrophic stimulus of abdominal aortic constriction or norepinephrine infusion, display TGF-β1 hyperexpression (42). On the other hand, prevention of myocardial hypertrophy with angiotensin-converting enzyme inhibitors is associated with decreased TGF-β1 expression (43).

A well-documented property of TGF-β1 is its ability to induce fibrosis and promote accumulation of extracellular matrix (9, 10, 44). TGF-β1-induced fibroblast proliferation, stimulation of collagen synthesis, and enhanced matrix accumulation contribute to the development of vascular disease as well as progressive renal disease.

Direct evidence that TGF-β1 expression can engender renal disease exists. In a rat model of glomerulonephritis, intrarenal expression of TGF-β1 was associated with increased extracellular matrix and anti-TGF-β1 antibodies prevented matrix assembly (44). In a diabetic mouse model, anti-TGF-β1 antibodies constrained TGF-β1 mRNA hyperexpression and prevented glomerular hypertrophy (45). In vivo transfection of the TGF-β1 gene resulted in glomerulosclerosis (46), and a TGF-β1 antisense oligomer reduced intrarenal expression of TGF-β1 and suppressed extracellular matrix accumulation (47). Of particular relevance to our observations of higher levels of TGF-β1 in black hypertensives, high-circulating levels of TGF-β1 protein have been reported to lead to fibrotic disease of kidneys and other organs in an experimental glomerulonephritis model. In TGF-β1 transgenic mice, high-circulating levels were associated with mesangial expansion, glomerular immune deposits, extracellular matrix accumulation, interstitial fibrosis, and progressive renal disease (15). Indeed, 25% of the transgenic mice died of renal failure.

Increased Frequency of TGF-β1 Hyperexpression as a Pathogenetic Mechanism for Excess Hypertension in Blacks.

Genetic as well as socioenvironmental factors have been proposed to explain the heightened incidence of hypertension and hypertensive complications in blacks, and both are likely to be important. Polymorphisms in several candidate genes for hypertension such as angiotensinogen, angiotensin-converting enzyme, α2-adrenergic receptor, and polymorphisms in genes coding mitochondrial proteins all have been reported to be more frequent in blacks compared with whites (48). Social, psychological, and cultural variances also have been explored, but to date, there is no consensus regarding the relative importance of any of these factors.

We hypothesize that TGF-β1 hyperexpression is a pathogenetic mechanism for the excess burden of hypertension and hypertensive complications in blacks compared with whites. The biological attributes of TGF-β1 and the reported association between TGF-β1 overexpression and cardiovascular and renal pathology support the postulate that the increased frequency of TGF-β1 overexpression in blacks is a candidate mechanism for hypertension and hypertensive complications, particularly the progression of hypertensive renal disease to ESRD.

An intriguing aspect of hypertension and progression to renal failure in blacks is that aggressive blood pressure control does not always lead to similar renal benefits in blacks and whites. Rostand et al. (7) have reported that renal function deteriorated more rapidly in hypertensive black patients compared with hypertensive whites despite similar levels of blood pressure control. Perry et al. (8) have noted a 2-fold higher risk of ESRD among black hypertensives compared with their white counterparts. The Multiple Risk Factor Intervention Trial study has shown that for every level of blood pressure control, blacks are at an increased risk for the development of renal disease compared with whites with the same blood pressure (6). These observations together suggest that other risk factor(s), in addition to hypertension, contribute to hypertension-associated renal disease in blacks.

In view of the experimental evidence that TGF-β1 hyperexpression results in renal disease, and in light of our data, we suggest that the increased frequency of TGF-β1 hyperexpression contributes to the excess burden of ESRD in blacks and, particularly, to ESRD attributed to hypertensive disease.

In summary, our cross-sectional studies have identified that TGF-β1 is overexpressed in hypertensive subjects compared with normotensive controls and that hyperexpression is more prevalent in blacks compared with whites. The protean biological activities of TGF-β1 and the experimental and clinical data suggest that TGF-β1 hyperexpression is a candidate mechanism for hypertension. Our observations support the idea that the heightened frequency of TGF-β1 overexpression in blacks contributes to the excess burden of hypertension and hypertensive complications in blacks. Further exploration of the contribution of TGF-β1 to hypertension and hypertensive complications in a prospective investigation might provide new insights into mechanisms of hypertension as well as suggest novel therapeutic targets.

Acknowledgments

We are grateful to Ms. G. Helseth for her diligent efforts toward sample collection and Ms. L. Stackhouse for meticulous assistance in the preparation of this manuscript. This work was supported in part by an award from the National Institutes of Health (R01 DK54943).

Abbreviations

ESRD

end-stage renal disease

TGF-β1

transforming growth factor-β1

PBMC

peripheral blood mononuclear cells

ARMS-PCR

amplification-refractory mutation system–PCR

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.050420897.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.050420897

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