Patricia I. Porto

Antisense Inhibition of Thyrotropin-Releasing Hormone Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.

Thyrotropin-releasing hormone receptor (TRHR) gene is associated with essential hypertension

In essential hypertension, a polygenic and multifactorial syndrome, several genes interact with the environment to produce high blood pressure. Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. We have described that TRH overexpression induces hypertension in a normal rat, which was reversed by TRH antisense treatment. This treatment also reduces the central TRH hyperactivity in spontaneously hypertensive rats and normalizes blood pressure. Human TRH receptor (TRHR) belongs to the G protein-coupled seven-transmembrane domain receptor superfamily. Mutations of these receptors may result in constitutive activation. As it has been demonstrated that hypertensive patients have a blunted TSH response to TRH injection, suggesting a defect in the TRHR, we postulate that the TRHR gene is involved in human hypertension. We studied 2 independent populations from different geographic regions of our country: a sample of adult subjects from a referral clinic and a population-based sample of high school students. In search of molecular variants of TRHR, we disclosed that a polymorphic TG dinucleotide repeat (STR) at −68 bp and a novel single nucleotide polymorphism, a G→C conversion at −221 located in the promoter of the TRHR are associated with essential hypertension. As STRs detected in gene promoters are potential Z-DNA-forming sequences and seem to affect gene expression, we studied the potentially different transcriptional activity of these TRHR promoter variants and found that the S/−221C allele has a higher affinity than does the L/G−221 allele to nuclear protein factor(s). Our findings support the hypothesis that the TRHR gene participates in the etiopathogenesis of essential hypertension.

Clinical features of the metabolic syndrome in adolescents: Minor role of the Trp64Arg β3-Adrenergic receptor gene variant

Obesity and hypertension are increasing medical problems in adolescents. We evaluated the association between being overweight—particularly abdominal fat—and having hypertension and assessed the contribution of the Trp64Arg β3-adrenergic receptor gene variant. In a population-based study, we determined family history, anthropometric variables, and arterial blood pressure of 934 high school students, out of whom we selected 121 normotensive and 54 hypertensive students. Biochemical measurements included circulating renin and angiotensin-converting enzyme activities, leptin, glucose, insulin and lipid levels, and β3-adrenergic receptor genotypes. We used Mann-Whitney U test, χ2-test, and Spearman rank-order correlation. In the total population, hypertension prevalence increased across the entire range of body mass index (BMI) percentiles. In the sample, hypertensive students showed higher BMI, waist-to-hip ratio, triglycerides, and insulin resistance and lower HDL-cholesterol than normotensive students did. Age- and sex-adjusted systolic arterial blood pressure was correlated with BMI, waist-to-hip ratio, insulin resistance, and leptin. Leptin was correlated with BMI and homeostasis model assessment method. We found no association among hypertension, BMI, and leptin levels with β3-adrenergic receptor genotypes. Especially in girls, the waist-to-hip ratio was, however, suggestively higher in Arg64 variant carriers than in noncarriers, independent of hypertension. In fact, there was a significantly (p < 0.01) higher frequency of carriers of the Arg64 variant across the waist-to-hip ratio quartiles. In adolescents of European origin, hypertension is associated with an increased degree of obesity among other characteristics of the metabolic syndrome; the Trp64Arg variant of the β 3-adrenergic receptor gene may favor the central adiposity gain.

Renin–Angiotensin–Aldosterone System Loci and Multilocus Interactions in Young‐Onset Essential Hypertension

Objective: Renin–angiotensin–aldosterone system component genes have been associated to essential hypertension. Thus, we studied the association of singe locus or multilocus interactions with young‐onset essential hypertension. Setting and Design: This is a case‐control study based on a population sample of adolescent at an inner city. Participants: We studied 54 adolescents with hypertension and 121 age‐matched normotensives, recruited from a high‐school student population of 934 interviewed individuals. Methods: Resting blood pressure was measured on three different days and normalized (Z‐score) by sex and age. Genotypes of ACE (I/D) angiotensinogen (T174M and M235T), AT1R (A1166C), and CYP11B2 (C‐344T) were determined by PCR/RFLP or ASO. Results: Although genotype frequencies were not different in both groups, we found a significant dominant effect of ACE D and angiotensinogen 235T alleles on normalized systolic arterial blood pressure in males. This effect was confirmed by sib‐pair linkage analysis taking normalized blood pressure as a quantitative trait. We independently analyzed multilocus interactions in normotensive and hypertensive adolescents searching for multiple locus deviation from Hardy–Weinberg or linkage equilibrium. We found that from 63 multilocus combinations, 4 deviated significantly from equilibrium in hypertensive adolescents but none in the normotensives. Deviations from equilibrium may indicate that the combination of alleles at different loci affects susceptibility or resistance to the disease. Conclusion: In addition to the angiotensin‐converting enzyme (ACE) and angiotensinogen (AGT) gene variants, gene–gene interactions may be important causative factors in a complex disease such as young‐onset essential hypertension.

Hyperhomocysteinemia but not MTHFR genotype is associated with young-onset essential hypertension

Hyperhomocysteinemia but not MTHFR genotype is associated with young-onset essential hypertension

Central overexpression of the TRH precursor gene induces hypertension in rats: antisense reversal.

Extrahypothalamic TRH participates in cardiovascular regulation and spontaneous hypertension of the rat. To investigate whether an increase in central TRH activity produces hypertension we studied the effect of the preTRH overproduction induced by I.C.V. transfection with a naked eukaryotic expression plasmid vector which encodes preTRH (pCMV-TRH). Northern blot analysis and RT-PCR showed that pCMV-TRH was transcribed in vitro and in vivo. At 24, 48, and 72 hours, pCMV-TRH (100 microg) in a significant and dose-dependent manner increased 37%, 84%, and 49%, respectively, the diencephalic TRH content and SABP (42+/-3, 50+/-2, and 22+/-2 mm Hg, respectively) with respect to the vector without the preTRH cDNA insert (V[TRH(-)]) as measured by RIA and the plethysmographic method, respectively, in awake animals. In addition, using immunohistochemistry we found that the increase of TRH was produced in circumventricular areas where the tripeptide is normally located. To further analyze the specificity of these effects we studied the actions of 23-mer sense (S), antisense (AS), and 3self-stabilized sense (Ss) and antisense (ASs) phosphorothioate oligonucleotides against the initiation codon region. Only ASs inhibited the increase of TRH content and SABP induced by pCMV-TRH treatment. In addition, pCMV-TRH-induced hypertension seems not to be mediated by central Ang II or serum TSH. To summarize, central TRH overproduction in periventricular areas induced by I.C.V. transfection produces hypertension in rats which is reversed by specific antisense treatment. This model may help in testing effective antisense oligodeoxynucleotides against other candidate genes.

Central Antisense Treatment Against Thyroliberin Normalizes Blood Pressure in Spontaneously Hypertensive Rats Without Affecting Thyroid Status.

P202 Thyroliberin (TRH) participates in central cardiovascular regulation and hypothalamic TRH precursor gene overexpression induces hypertension which can be reversed by antisense (AS) treatment. SHR show an increased central TRH synthesis and release and TRH receptor number. We show here that a phosphotioate AS against the preTRH injected icv decreased the augmented diencephalic TRH content (ng/mg protein) along with a normalization of systolic blood pressure (SABP) at 24-48 hs in SHR without effect in normotensive WKY rats (table). Saline or sense treatment had no effects. To investigate if changes in thyroid function may explain these results, we studied plasma TSH, T 3 (ng/ml) and T 4 (ug%) in control (CON) and AS-treated WKY and SHR. Although TSH was elevated in SHR compared to WKY rats in basal conditions and AS reverted it (table), no differences (n=5) were observed between strains with or without AS treatment in plasma T 4 (CON; WKY: 5.0±0.4 vs SHR: 5.3±0.5 and AS; WKY:5.4±0.3 vs SHR: 5.6±0.4) or T 3 (CON; WKY: 1.3±0.4 vs SHR: 1.1±0.2 and AS; WKY: 1.5±0.2 vs SHR: 1.4±0.3). To summarize, TRH AS decreases the higher diencephalic TRH content and changed arterial blood pressure to that of WKY rats even though SHR were basally and after AS treatment remained clinically euthyroid. These results suggest that the central TRH system is involved in the etiopathogenesis of hypertension in this model.