Valproate decreases transgenerationally blood pressure by affecting thyrotropin-releasing hormone promoter DNA methylation and gene expression in spontaneously hypertensive rat.

Central TRH, a neuropeptide, is involved in cardiovascular regulation. We demonstrated that the overexpression of diencephalic TRH (dTRH) in SHR rats can be prevented by antisense treatment, normalizing blood pressure (BP). Valproate (VPA) is an inhibitor of histone deacetylases (HDAC) which modulates gene expression through epigenetic modifications such as DNA methylation. AIMS: Study the role of HDAC inhibition in the regulation of dTRH gene expression and its effect on the pathogenesis of hypertension. MAIN METHODS: We treated 7-weeks-old male and female SHR and WKY rats with VPA for 10 weeks and evaluated BP, dTRH mRNA and methylation gene status. KEY FINDINGS: VPA attenuated the elevated BP and dTRH mRNA expression characteristic of SHR. Indeed, we found a significant 62% reduction in dTRH mRNA expression in the SHR + VPA group compared to control SHR. The decrease TRH mRNA expression induced by VPA was confirmed "in vitro" in a primary neuron culture using trichostatin A. With methylation specific PCR we demonstrated a significant increase in TRH promoter DNA methylation level in SHR + VPA group compared to control SHR. After 2 weeks of treatment interruption, rats were mated. Although they did not receive any treatment, the offspring born from VPA-treated SHR parents showed similar changes in BP, dTRH expression and methylation status, implying a transgenerational inheritance. Our findings suggest that dTRH modulation by epigenetics mechanism affects BP and could be inherited by the next generation in SHR rats.

Novel Leptin-Cardiac TRH pathway responsible for the cardiac alterations in the Hyperleptinemic obesity.

The association between hypertension and obesity-induced cardiac damage is usually accepted. However, no studies have been focused on cardiac alterations in obesity, independently of blood pressure increase. It is well known that Cardiac TRH induces Left Ventricular Hypertrophy (LVH) and fibrosis, and its inhibition prevents the development of hypertrophy. Also, it has been described that the adiponectin leptin induces TRH expression. Thus, we hypothesized that in obesity, the increase in TRH induced by hyperleptinemia is responsible for LVH, until now mostly attributed to pressure load. We studied obese Agouti mice suffering from hypertension with hyperleptinemia and found a significant LVH development with increased TRH gene expression. Consequently, we found higher fibrotic (collagens and TGF-ß) and hypertrophic markers (BNP and ß-MHC) expression vs lean black controls. As pressure could explain these results, we treated obese mice with diuretic (hydrochlorothiazide 20 mg/kg/day) since weaning. Diuretic treatment was successful as the diuretic group was normotensive in contrast to control obese mice. Nevertheless, both groups showed LVH development, higher cardiac precursor TRH gene and peptide expressions and elevated fibrotic and hypertrophic markers expression, pointing out that obesity-induced LVH is not due to hypertension. In addition, we performed Cardiac TRH inhibition by specific siRNA injection compared to control siRNA treatment and evaluated cardiac damage. As expected, expressions and protein increase in hypertrophic and fibrotic markers observed in the AG mouse with the native cTRH system were not seen in the AG mouse with the cTRH silencing. Indeed, the AG + TRH-siRNA group showed hypertrophic markers expression and fibrosis measurements similar to the lean BL mice. On the whole, these results point out that the novel Leptin-Cardiac TRH pathway is responsible for the cardiac alterations present in hyperleptinemic obesity, independent of blood pressure, and cTRH long-term silencing since early stages totally prevent LVH development and cardiac fibrosis.

Cardiac Thyrotropin-releasing Hormone Inhibition Improves Ventricular Function and Reduces Hypertrophy and Fibrosis After Myocardial Infarction in Rats.

BACKGROUND: Cardiac thyrotropin-releasing hormone (TRH) is a tripeptide with still unknown functions. We demonstrated that the left ventricle (LV) TRH system is hyperactivated in spontaneously hypertensive rats and its inhibition prevented cardiac hypertrophy and fibrosis. Therefore, we evaluated whether in vivo cardiac TRH inhibition could improve myocardial function and attenuate ventricular remodeling in a rat model of myocardial infarction (MI). METHODS AND RESULTS: In Wistar rats, MI was induced by a permanent left anterior descending coronary artery ligation. A coronary injection of a specific small interfering RNA against TRH was applied simultaneously. The control group received a scrambled small interfering RNA. Cardiac remodeling variables were evaluated one week later. In MI rats, TRH inhibition decreased LV end-diastolic (1.049 ± 0.102 mL vs 1.339 ± 0.102 mL, P < .05), and end-systolic volumes (0.282 ± 0.043 mL vs 0.515 ± 0.037 mL, P < .001), and increased LV ejection fraction (71.89 ± 2.80% vs 65.69 ± 2.85%, P < .05). Although both MI groups presented similar infarct size, small interfering RNA against TRH treatment attenuated the cardiac hypertrophy index and myocardial interstitial collagen deposition in the peri-infarct myocardium. These effects were accompanied by attenuation in the rise of transforming growth factor-ß, collagen I, and collagen III, as well as the fetal genes (atrial natriuretic peptide, B-type natriuretic peptide, and beta myosin heavy chain) expression in the peri-infarct region. In addition, the expression of Hif1α and vascular endothelial growth factor significantly increased compared with all groups. CONCLUSIONS: Cardiac TRH inhibition improves LV systolic function and attenuates ventricular remodeling after MI. These novel findings support the idea that TRH inhibition may serve as a new therapeutic strategy against the progression of heart failure.

Cardiovascular and body weight regulation changes in transgenic mice overexpressing thyrotropin-releasing hormone (TRH).

Thyrotropin-releasing hormone (TRH) plays several roles as a hormone/neuropeptide. Diencephalic TRH (dTRH) participates in the regulation of blood pressure in diverse animal models, independently of the thyroid status. The present study aimed to evaluate whether chronic overexpression of TRH in mice affects cardiovascular and metabolic variables. We developed a transgenic (TG) mouse model that overexpresses dTrh. Despite having higher food consumption and water intake, TG mice showed significantly lower body weight respect to controls. Also, TG mice presented higher blood pressure, heart rate, and locomotor activity independently of thyroid hormone levels. These results and the higher urine noradrenaline excretion observed in TG mice suggest a higher metabolic rate mediated by sympathetic overflow. Cardiovascular changes were impeded by siRNA inhibition of the diencephalic Trh overexpression. Also, the silencing of dTRH in the TG mice normalized urine noradrenaline excretion, supporting the view that the cardiovascular effects of TRH involve the sympathetic system. Overall, we show that congenital dTrh overexpression leads to an increase in blood pressure accompanied by changes in body weight and food consumption mediated by a higher sympathetic overflow. These results provide new evidence confirming the participation of TRH in cardiovascular and body weight regulation.

Short-term doxorubicin cardiotoxic effects: involvement of cardiac Thyrotropin Releasing Hormone system.

Doxorubicin is an antineoplastic in the anthracycline class widely used for the treatment of several solid tumors and blood cancers. Cardiotoxicity is the major dose-limiting adverse effect of the drug. Chronic and accumulated doxorubicin administration cause myocyte damage and myocardial fibrosis. Doxorubicin-associated cardiotoxicity can be also observed after a short-course drug treatment even without clinical evidence of cardiac disease. Nevertheless, acute underlying mechanisms involved in the initiation of drug-induced cardiotoxicity remain poorly explored despite their similarities with pathophysiological conditions where cardiac TRH (cTRH) plays a central role. We showed that cTRH mediates myocardial injury induced by hypertension, and angiotensin II. Further, cTRH overexpression induces cardiac apoptosis, hypertrophy and fibrosis. AIM: To demonstrate that cTRH could mediate acute doxorubicin cardiotoxicity. MAIN METHOD: A single injection of doxorubicin (10 mg kg/day i.p.) was used to evaluate acute cardiac damage in a short-term experimental model of doxorubicin-induced cardiotoxicity. While inhibiting cTRH by small interfering RNA (siRNA), we evaluated the progression of cardiotoxicity. KEY FINDINGS: We found a doxorubicin-induced TRH overexpression in the LV, which was associated with apoptosis, hypertrophy and fibrosis. siRNA-mediated cTRH suppression prevented the doxorubicin-associated cardiac histological lesions. SIGNIFICANCES: doxorubicin requires an active cardiac TRH system to promote heart injury.

Angiotensin II requires an intact cardiac thyrotropin-releasing hormone (TRH) system to induce cardiac hypertrophy in mouse.

Cardiac tyhrotropin-releasing hormone (TRH) is overexpressed in the hypertrophied left ventricle (LV) of spontaneously hypertensive rats (SHR) and its inhibition prevents both hypertrophy and fibrosis. In a normal heart, the TRH increase induces fibrosis and hypertrophy opening the question of whether TRH could be a common mediator of left ventricular hypertrophy (LVH). We used angiotensin II (AngII) as an inductor of LVH to evaluate if the blockade of LV-TRH prevents hypertrophy and fibrosis in mice. We challenged C57BL/6 adult male mice with an infusion of AngII (osmotic pumps; 2 mg/kg.day) to induce LVH. Groups of mice were injected with an intracardiac siRNA-TRH or scrambled siRNA (siRNA-Con). Body weight, water intake and systolic arterial blood pressure (SABP) were measured daily. AngII significantly increased water intake and SABP (p < .05). Cardiac hypertrophy (heart weight/body weight) was evident in the group with the normal cardiac TRH system. In fact, it was found an AngII-induced increase of TRH precursor mRNA (p < .05) in conjunction with elevated TRH levels measured by immunohistochemistry and western blot. These changes were not observed in the AngII + siRNA-TRH group. Furthermore, AngII increased significantly (p < .05) BNP (hypertrophic marker), collagens I and III and TGF-ß (fibrosis markers) expression in the group with the native cardiac TRH system. These increases were attenuated in the groups with the TRH system blocked despite the high blood pressure. Similar and stronger results were observed "in vitro" with NIH3T3 and H9C2 cell culture models, where, when the TRH system is blocked, AngII stimulus was not able to induce the markers of its fibrotic and hypertrophic effects, so we believe that these effects are independent of any other physiological modifications. Our results point out that cardiac TRH is required for AngII-induced hypertrophic and fibrotic effects.

Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis.

Deficiency of the antiaging gene Klotho (KL) induces renal damage and hypertension through unknown mechanisms. In this study, we assessed whether KL regulates expression of CYP11B2, a key rate-limiting enzyme in aldosterone synthesis, in adrenal glands. We found that haplodeficiency of KL(+/-) in mice increased the plasma level of aldosterone by 16 weeks of age, which coincided with spontaneous and persistent elevation of BP. Blockade of aldosterone actions by eplerenone reversed KL deficiency-induced hypertension and attenuated the kidney damage. Protein expression of CYP11B2 was upregulated in adrenal cortex of KL(+/-) mice. KL and CYP11B2 proteins colocalized in adrenal zona glomerulosa cells. Silencing of KL upregulated and overexpression of KL downregulated CYP11B2 expression in human adrenocortical cells. Notably, silencing of KL decreased expression of SF-1, a negative transcription factor of CYP11B2, but increased phosphorylation of ATF2, a positive transcription factor of CYP11B2, which may contribute to upregulation of CYP11B2 expression. Therefore, these results show that KL regulates adrenal CYP11B2 expression. KL deficiency-induced spontaneous hypertension and kidney damage may be partially attributed to the upregulation of CYP11B2 expression and aldosterone synthesis.

Thyrotropin-releasing hormone overexpression induces structural changes of the left ventricle in the normal rat heart.

Thyrotropin-releasing hormone (TRH) hyperactivity has been observed in the left ventricle of spontaneously hypertensive rats. Its long-term inhibition suppresses the development of hypertrophy, specifically preventing fibrosis. The presence of diverse systemic abnormalities in spontaneously hypertensive rat hearts has raised the question of whether specific TRH overexpression might be capable of inducing structural changes in favor of the hypertrophic phenotype in normal rat hearts. We produced TRH overexpression in normal rats by injecting into their left ventricular wall a plasmid driving expression of the preproTRH gene (PCMV-TRH). TRH content and expression of preproTRH, collagen type III, brain natriuretic peptide, ß-myosin heavy chain, Bax-to-Bcl-2 ratio, and caspase-3 were measured. The overexpression maneuver was a success, as we found a significant increase in both tripeptide and preproTRH mRNA levels in the PCMV-TRH group compared with the control group. Immunohistochemical staining against TRH showed markedly positive brown signals only in the PCMV-TRH group. TRH overexpression induced a significant increase in fibrosis, evident in the increase of collagen type III expression accompanied by a significant increase in extracellular matrix expansion. We found a significant increase in brain natriuretic peptide and ß-myosin heavy chain expression (recognized markers of hypertrophy). Moreover, TRH overexpression induced a slight but significant increase in myocyte diameter, indicating the onset of cell hypertrophy. We confirmed the data "in vitro" using primary cardiac cell cultures (fibroblasts and myocytes). In conclusion, these results show that a specific TRH increase in the left ventricle induced structural changes in the normal heart, thus making the cardiac TRH system a promising therapeutic target.

Cardiac thyrotropin-releasing hormone mediates left ventricular hypertrophy in spontaneously hypertensive rats.

Local thyrotropin-releasing hormone (TRH) may be involved in cardiac pathophysiology, but its role in left ventricular hypertrophy (LVH) is still unknown. We studied whether local TRH is involved in LVH of spontaneously hypertensive rats (SHR) by investigating TRH expression and its long-term inhibition by interference RNA (TRH-iRNA) during LVH development at 2 stages (prehypertrophy and hypertrophy). SHR and their control rats (WKY) were compared. Cardiac hypertrophy was expressed as heart/total body weight (HW/BW) ratio. TRH content (radioimmuno assay), preproTRH, TRH receptor type I, brain natriuretic peptide (BNP), and collagen mRNA expressions (real-time polymerase chain reaction) were measured. For long-term inhibition of TRH, TRH-iRNA was injected into the left ventricle (LV) wall for 8 weeks. Hearts were processed for morphometric studies and immunohistochemical analysis using antibodies against α-smooth muscle actin and collagen type III. LV preproTRH-mRNA abundance was similar in both strains at 7 weeks of age. At the hypertrophic stage (18 weeks old), however, there was a 15-fold increase in SHR versus WKY, consistent with a significant increase in tripeptide levels and the expression of its receptor. Specific LV-TRH inhibition at the prehypertensive stage with TRH-iRNA, which decreased >50% preproTRH expression and tripeptide levels, prevented LVH development as shown by the normal HW/BW ratio observed in TRH-iRNA-treated SHR. In addition, TRH-iRNA impeded the increase in BNP and type III collagen expressions and prevented the increase in cardiomyocyte diameter evident in mismatch iRNA-treated adult SHR. These results show for the first time that the cardiac TRH system is involved in the development of LVH in SHR.

Gene prioritization based on biological plausibility over genome wide association studies renders new loci associated with type 2 diabetes.

PURPOSE: We present an approach to prioritize single nucleotide polymorphisms for further follow-up in genome-wide association studies of type 2 diabetes. METHOD: The proposed method combines both the use of open data access from two type 2 diabetes-genome-wide association studies (granted by the Diabetes Genetics Initiative and the Welcome Trust Case Control Consortium) and the comprehensive analysis of candidate regions generated by the freely accessible ENDEAVOUR software. RESULTS: The algorithm prioritized all genes of the whole genome in relation to type 2 diabetes. There were six of 1096 single nucleotide polymorphisms in five genes potentially associated with type 2 diabetes: tachykinin receptor 3 (rs1384401), anaplastic lymphoma receptor tyrosine kinase (rs4319896), calcium channel, voltage-dependent, L type, alpha 1D subunit (rs12487452), FOXO1A (rs10507486 and rs7323267), and v-akt murine thymoma viral oncogene homolog 3 (rs897959). We estimated the fixed effect and P values of each single nucleotide polymorphism in the combined dataset by Mantel-Haenszel meta-analysis and we observed significant P values for all single nucleotide polymorphisms except for rs897959 at v-akt murine thymoma viral oncogene homolog 3. CONCLUSION: The proposed strategy may be used as an alternative tool for optimizing the information of the nearly 500,000 gene variants in which markers with modest significant P value for disease association are currently disregarded. Additionally, the said single nucleotide polymorphisms may be incorporated into the replication of the multistage design involved in the genome-wide association studies.

Losartan reduces liver expression of plasminogen activator inhibitor-1 (PAI-1) in a high fat-induced rat nonalcoholic fatty liver disease model.

OBJECTIVE: To evaluate the effect of losartan-an angiotensin II type 1 receptor (AT1R) antagonist- and telmisartan-an AT1R blocker with insulin-sensitizing properties-, on the hepatic expression of plasminogen activator inhibitor-1 (PAI-1) in a rat model of nonalcoholic fatty liver disease (NAFLD). METHODS: Rats were given a high-fat diet (HFD) for 8 weeks and after this period were randomly divided into 3 groups. For 12 weeks along with the same access to HFD, one group (9 rats) received losartan and another group received telmisartan (10 rats), both at 10mg/kg intraperitoneally (ip) every 24h. The third group (8 rats) received saline ip along with the HFD. Finally, a control group (6 rats) was fed with standard chow diet for 20 weeks. RESULTS: Fatty liver was reverted by both losartan and telmisartan. Both drugs had beneficial effects on insulin resistance, reaching statistical significance in telmisartan group. Expression of hepatic mRNA of PAI-1 showed a 42% decrease in losartan-treated rats in comparison with both HFD group and telmisartan-treated rats. To further evaluate this differential effect on PAI-1 expression, we explored the effect of the drugs on liver expression of TNFalpha, PEPCK-C and PPARalpha, and no significant differences were observed. CONCLUSION: These results indicate that AT1R blockers could be eligible drugs for reducing hepatic lipid accumulation in patients with NAFLD. However, only 12 weeks of losartan treatment strongly reduced hepatic PAI-1 gene expression. These differences could provide even more effective options for preventing fatty liver disease and its cardiovascular complications.

Involvement of angiotensin-(1-7) in the hypothalamic hypotensive effect of captopril in sinoaortic denervated rats.

The role of anterior hypothalamic angiotensin-(1-7) (Ang-(1-7)) on blood pressure regulation was studied in sinoaortic denervated (SAD) rats. Since angiotensin-converting enzyme inhibitors increase endogenous levels of Ang-(1-7), we addressed the involvement of Ang-(1-7) in the hypotensive effect induced by captopril in SAD rats. Wistar rats 7 days after SAD or sham operation (SO) were anaesthetized and the carotid artery was cannulated for monitoring mean arterial pressure (MAP). A needle was inserted into the anterior hypothalamus for drug administration. Intrahypothalamic administration of Ang-(1-7) (5 pmol) was without effect in SO rats but reduced MAP in SAD rats by 15.5+/-3.2 mm Hg and this effect was blocked by 250 pmol [D-Ala(7)]-Ang-(1-7), a Mas receptor antagonist. Angiotensin II (Ang II) induced an increase in MAP in both groups being the effect greater in SAD rats (DeltaMAP=15.8+/-1.4 mm Hg) than in SO rats (DeltaMAP=9.6+/-1.0 mm Hg). Ang-(1-7) partially abolished the pressor response caused by Ang II in SAD rats. Whilst the captopril intrahypothalamic injection did not affect MAP in SO animals, it significantly reduced MAP in SAD rats (DeltaMAP=-13.3+/-1.9 mm Hg). Either [D-Ala(7)]-Ang-(1-7) or an anti-Ang-(1-7) polyclonal antibody partially blocked the MAP reduction caused by captopril. In conclusion, whilst Ang-(1-7) does not contribute to hypothalamic blood pressure regulation in SO normotensive animals, in SAD rats the heptapeptide induces a reduction of blood pressure mediated by Mas receptor activation. Although Ang-(1-7) is not formed in enough amount in the AHA of SAD animals to exert cardiovascular effects in normal conditions, our results suggest that enhancement of hypothalamic Ang-(1-7) levels by administration of captopril is partially involved in the hypotensive effect of the ACE inhibitor.

Association between diencephalic thyroliberin and arterial blood pressure in agouti-yellow and ob/ob mice may be mediated by leptin.

Leptin, a hormone secreted by the adipose tissue, stimulates anorexigenic peptides and also inhibits orexigenic peptides in hypothalamic arcuate nuclei-located neurons. It also counteracts the starvation-induced suppression of thyroid hormones by up-regulating the expression of preproTRH gene. On the other hand, in addition to its role as a modulator of the thyroid-hypothalamic-hypophysial axis, thyrotropin-releasing hormone (TRH) acts as a modulator of the cardiovascular system. In fact, we reported that overexpression of diencephalic TRH (dTRH) induces hypertension. We have recently shown that, in rats with obesity-induced hypertension, hyperleptinemia may produce an increase of dTRH together with an elevation of arterial blood pressure (ABP) through an increase of sympathetic activity and that these alterations were reversed by antisense oligonucleotide and small interfering RNA against preproTRH treatments. Here we explore the possible role of dTRH as a mediator involved in leptin-induced hypertension in 2 obesity mouse models: agouti-yellow mice, which are hyperleptinemic and hypertensive, and ob/ob mice, which lack functional circulating leptin. These 2 models share some characteristics, but ob/ob mice show lower ABP and plasma catecholamines levels. Then, for the first time, we report that there is a clear association between ABP and dTRH levels in both mouse models, as we have found that dTRH content was elevated in agouti-yellow mice and diminished in ob/ob mice compared with their controls. We also show that, after 3 days of subcutaneous leptin injections (10 microg/12 hours), ABP and dTRH increased significantly in ob/ob mice with no alterations of thyroid hormone levels. These results add evidence to the putative molecular mechanisms for the strong association between obesity and hypertension.

SiRNA-mediated silencing of the diencephalic thyrotropin-releasing hormone precursor gene decreases the arterial blood pressure in the obese agouti mice.

Obesity is associated with increased cardiovascular morbidity and mortality, in part through development of hypertension. Leptin promotes weight loss by reducing food intake and increasing energy expenditure through sympathetic stimulation. It also counteracts the starvation-induced suppression of thyroid hormone by up-regulating the expression of TRH. On the other hand, it is known that the extrahypothalamic TRH system participates in cardiovascular function modulating sympathetic system activity. In order to challenge the testable hypothesis that obesity may raise arterial blood pressure (ABP) through TRH system activation, we herein analyze the participation of the TRH system in the elevation of ABP in the obese agouti yellow mice. These mice are characterized by resistance to the weight reducing effect of leptin although they show a preserved sympathetic response to leptin along with a mild hypertension compared with the control strain (121+/-2 vs 102+/-2 mmHg, p less than 0.001, n=10). We report here that hyperleptinemic agouti mice showed a 1.8-fold elevation of diencephalic TRH content compared with controls, and we demonstrate that a long lasting specific inhibition of TRH system by icv treatment with siRNA against preproTRH normalizes systolic ABP independently of the thyroid status. These results suggest that the interaction leptin-diencephalic TRH may be one of the mechanisms involved in the mild hypertension of the obese agouti mice.

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat.

We recently showed that diencephalic TRH may mediate the central leptin-induced pressor effect. Here, to study the role of TRH in obesity-induced hypertension (OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP) increased in HFD animals. Plasma leptin was correlated with peritoneal adipose tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and small interfering (si)RNA against the prepro-TRH. Antisense significantly decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment. Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk. Conversely, vehicle, an inverted antisense sequence and siRNA against green fluorescence protein, produced no changes. SABP decrease seems to be owing to an inhibition of the obesity-enhanced sympathetic outflow but not to an alteration in thyroid status. Using a simple OIH model we demonstrated, for the first time, that central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity. Thus the TRH-leptin interaction may contribute to the strong association between hypertension and obesity.

Integrative study of hypothalamus-pituitary-thyroid-immune system interaction: thyroid hormone-mediated modulation of lymphocyte activity through the protein kinase C signaling pathway.

Thyroid hormones play critical roles in differentiation, growth and metabolism, but their participation in immune system regulation has not been completely elucidated. Modulation of in vivo thyroid status was used to carry out an integrative analysis of the role of the hypothalamus-pituitary-thyroid (HPT) axis in T and B lymphocyte activity. The participation of the protein kinase C (PKC) signaling pathway and the release of some cytokines upon antigenic stimulation were analyzed. Lymphocytes from hyperthyroid mice displayed higher T-and B-cell mitogen-induced proliferation, and those from hypothyroid mice displayed lower T- and B-cell mitogen-induced proliferation, compared with euthyroid animals. Reversion of hypothyroid state by triiodothyronine (T3) administration recovered the proliferative responses. No differences were found in lymphoid subset balance. Both total PKC content and mitogen-induced PKC translocation were higher in T and B cells from hyperthyroid mice, and lower in cells from hypothyroid mice, compared with controls. Levels of thyroid-stimulating (TSH) and TSH-releasing (TRH) hormones were not directly related to lymphocyte proliferative responses. After immunization with sheep red blood cells (SRBCs) and re-stimulation, in vitro spleen cells from hyper- or hypothyroid mice showed, respectively, increased or decreased production of interleukin (IL)-2 and interferon (IFN)-gamma cytokines. Additionally, an increase in IL-6 and IFN-gamma levels was found in hyperthyroid cells after in vivo injection and in vitro re-stimulation with lipopolysaccharide (LPS). Our results show for the first time a thyroid hormone-mediated regulation of PKC content and of cytokine production in lymphocytes; this regulation could be involved in the altered responsiveness to mitogen-induced proliferation of T and B cells. The results also confirm the important role that these hormones play in regulating lymphocyte reactivity.

Parathyroid hormone-related protein overexpression decreases blood pressure in spontaneously hypertensive rats.

We have recently demonstrated that arterial PTHrP expression and cardiovascular responses to this protein are altered in SHR compared with normotensive animals, Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats. To investigate whether the slightly, but significantly decreased, aortic PTHrP gene expression observed in SHR, compared to that of normotensive animals, may play a causative role in the maintenance of the elevated arterial blood pressure (ABP) of the SHR, we transfected a hepatic lobe with a PTHrP expression vector in a sense and antisense orientation. At 24 and 48 hours, sense pSV2neo-ECE induced a significant five-fold increase in PTHrP mRNA abundance with respect to antisense pSV2neo-ECE and vehicle. This increment in the PTHrP mRNA induced by the sense PTHrP expression vector was totally inhibited by the co-administration of the antisense PTHrP expression vector. At the same time, we observed a significant decrease of mean ABP (MABP) in SHR transfected with the sense pSV2neo-ECE to similar values as those obtained in the normotensive strain. Neither antisense PTHrP expression vector nor vehicle had any significant effect in any strain. Again, the effect of the sense PTHrP expression vector on MABP was blocked by the simultaneous treatment with the antisense PTHrP expression vector. At 48 hours, the hypotensive effect of the sense pSV2neo-ECE in SHR was reverted by the i.v. bolus injection of a specific competitive PTHrP receptor antagonist such as Nle8,18,Tyr34-bPTH(3-34)amide. We propose that a defect of this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in SHR. This defect can be ameliorated by transfecting tissues with protein-exporting capabilities, such as the liver. Finally, our work adds additional data to a cumulative body of evidence suggesting that it might be possible to design an effective gene therapy to treat the common polygenic and multifactorial form of hypertension by increasing the activity of potent and physiological vasodilators.

Thyrotropin-releasing hormone decreases leptin and mediates the leptin-induced pressor effect.

Leptin, an adipocyte-released hormone, modifies food intake and energy expenditure regulating hypothalamic-pituitary-thyroid axis function. We previously reported that thyrotropin-releasing hormone (TRH) precursor gene overexpression induces hypertension in the normal rat and that spontaneously hypertensive rats have central TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In both models, intracerebroventricular antisense (AS) treatment against the TRH precursor produced a dose-dependent reduction of the increased diencephalic TRH content while normalizing high arterial blood pressure. In this article, we report that male Wistar rats that were made hypertensive by intracerebroventricular injection of a eucaryotic expression plasmid containing the pre-TRH cDNA showed decreased leptin plasma levels and that pre-TRH AS treatment reversed this phenomenon. In addition, male and female spontaneously hypertensive rats showed lower levels of circulating leptin than did sex-matched Wistar-Kyoto control rats. This difference also was abated by the pre-TRH AS treatment. Conversely, 20 microg ICV leptin induced a long-lasting pressor effect (18 +/- 5 mm Hg, n=6, P<0.01, >60 minutes) that was not observed in pre-TRH AS pretreated rats (2 +/- 3 mm Hg, n=6) but persisted in rats used as controls that were treated with inverted oligonucleotide (20 +/- 6 mm Hg, n=4, P<0.01). These data suggest that in rats with TRH-induced hypertension, leptin is decreased, inducing compensatory adiposity. We propose that because leptin produces central TRH synthesis and release, obesity may induce hypertension through TRH system activation and that the TRH-leptin interaction may thus contribute to the strong association between hypertension and obesity.