Blood Pressure In Transgenic Mice Research Articles

Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

Abstract W P232: High Sodium Diet Did Not Increase Peripheral Blood Pressure in a Transgenic Mouse Model of Alzheimer's Disease

Introduction: The impairment of Ca2+ homeostasis has been connected to the deleterious effect of abnormal beta-amyloid (Ab) on the calcium sensing receptors (CaSR) in Alzheimer’s disease (AD). Elevated extracellular Ca2+ can also induce relaxation of arteries and prevent the development of hypertension. Change in ionic strength reduces the effect of CaSR agonists, such as Ab, and regulates Ca2+ homeostasis. Na+ gradient also increase ionic strength and may regulate Ca2+ homeostasis through CaSR and Na+/Ca2+ antiporter exchange to affect blood pressure. In non-pathological conditions, the elevation of Na+ gradient through high-sodium diet is considered as a reason for imbalance of Na+/Ca2+, and one of the several causes of hypertension and cardiovascular complications. However, the effect of Na+ gradient on AD patients is unclear. The purpose of this study was to test the hypothesis that in AD the increase of Na+ gradient diminishes the Ab ability on activation of CaSR and does not induce the hypertension. Method: We examined the effect of Na+ load on the arterial blood pressure in double transgenic mice harboring Ab precursor protein (APPswe), and presenilin-1 (PSEN1) as mouse model of AD (10-20 weeks, 25-30g). Wild type littermates were used as controls. Four groups: Control + low sodium diet (n=6); Control + high sodium diet (n=12); APP + low sodium diet (n=12); APP + high sodium diet (n=12) were tested for both systolic arterial blood pressure (SAP) and diastolic arterial blood pressure (DAP). Mice were fed a low- (0.08% NaCl) or a high-sodium (4% NaCl) diet for 3 months. Heart rate and SAP and DAP were measured noninvasively using tail blood pressure measurement device after 3 months. Results: Compared with mice fed standard diet (0.08% NaCl), mice fed high sodium diet showed markedly elevated SAP and DAP (134±4.8 compared with 162±2.8 mmHg, and 114±5.0 compared with 137±20 mmHg) (p&lt;0.0001). There was no significant difference in both SAP and DAP between APP mice fed standard (0.08% NaCl) and high-sodium (4% NaCl) diet. SAP and DAP in elevated Aβ mouse were not affected by high dietary levels of sodium. Conclusion: Data from this AD model suggest that APP transgenic mice is resistant to high sodium-induced increase in systolic and diastolic blood pressure.

Variable transcriptional regulation of the human aldosterone synthase gene causes salt-dependent high blood pressure in transgenic mice.

Aldosterone, synthesized in the adrenal cortex by the enzyme CYP11B2, induces positive sodium balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the human CYP11B2 (hCYP11B2) gene to human hypertension. hCYP11B2 gene promoter has 3 single-nucleotide polymorphisms in linkage disequilibrium: T/A at -663, T/C at -470, and C/T at -344. Variants ACT occur together and form the haplotype-I (Hap-I), whereas variants TTC constitute Hap-II. We hypothesize that these single-nucleotide polymorphisms, when present together, will lead to haplotype-dependent differences in the transcriptional regulation of the hCYP11B2 gene and affect blood pressure regulation. We evaluated differences in tissue expression in vivo and consequential effects on blood pressure stemming from the 2 haplotypes. Novel transgenic mice with the hCYP11B2 gene, targeted to the mouse HPRT locus, with either Hap-II or Hap-I variant are used in this study. Our results show increased adrenal and renal expression of hCYP11B2 in transgenic mice with Hap-I when compared with mice with Hap-II. Importantly, we observed increased baseline blood pressure in Hap-I transgenic mice, an effect accentuated by a high-salt diet. Pathophysiological effects of elevated aldosterone were corroborated by our results showing upregulation of proinflammatory markers in renal tissues from the transgenic mice with Hap-I. These findings characterize the haplotype-dependent regulation of the hCYP11B2 gene where -344T serves as a reporter polymorphism and show that Hap-I leads to increased expression of hCYP11B2, with permissive effects on blood pressure and inflammatory milieu.

Abstract 089: A Genetic Variant of Human Aldosterone Synthase Gene Causes Salt-Dependent High Blood Pressure in Transgenic Mice

Aldosterone, synthesized by the enzyme CYP11B2, induces positive sodium-balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the hCYP11B2 gene to human hypertension. We have identified three SNPs, in linkage disequilibrium, in the hCYP11B2 gene: T/A at -663, T/C at -470 and C/T at -344. Variants ACT occur together and form the haplotype I (Hap I) while variants TTC constitute haplotype II (Hap II). We hypothesize that these SNPs, when present together, will lead to haplotype-dependent transcription of the hCYP11B2 gene, differentially increase aldosterone and affect blood pressure. To this end, novel transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype II or I variant are used in the study. ChIP assay, using anti-RNA pol II antibody, shows increased Pol II binding to the chromatin from Hap I TG mice in adrenal (2.8 fold higher, p&lt;0.05) and renal tissues (1.3 fold higher, p&lt;0.05) as compared to chromatin extracts from Hap II TG mice. Immunoblot analysis shows upregulation of the hCYP11B2 in adrenal (2.7 fold higher, p&lt;0.05) and renal tissues (1.35 fold higher, p&lt;0.05) of Hap I vs. Hap II-TG mice; no significant difference was observed in mCYP11B2 between the two haplotypes. Complementary ELISA shows higher circulating levels (p&lt;0.05) of aldosterone in Hap I mice (1504±48.7 pg/mL) as compared to both, Hap II (778±142.8 pg/mL) and C57 mice (740±28.9 pg/mL). Importantly, we observed increased baseline blood pressure in Hap I TG mice (Hap I- 117±2.5 vs. Hap II- 109±1.9 mm Hg, p&lt;0.05), an effect accentuated by high-salt diet (Hap I- 135±2.6 vs. Hap II- 122±2.2 mm Hg, p&lt;0.05). Elevated aldosterone was accompanied by up-regulation (p&lt;0.05) of proinflammatory markers in renal tissues from Hap I TG mice (IL1β, MCP1, ICAM). Thus, this study identifies -344T as a reporter polymorphism for Hap I of the hCYP11B2 gene. SNPs in Hap I promote increased transcription and expression of the gene, in multiple tissues, with resultant elevation of plasma aldosterone levels. Pathophysiological impact of this haplotype-dependent transcriptional regulation of the hCYP11B2 is highlighted by increased inflammation and blood pressure in TG mice with the Hap I of this transgene.

Abstract 626: Differential Regulation of Blood Pressure in Transgenic Mice Containing two Haplotypes of Human Angiotensin Receptor Type 1

The renin angiotensin system (RAS) is the main regulator of blood pressure and fluid homeostasis. Most of the known physiological and pathophysiological action of RAS are mediated through the angiotensin receptor type 1 (AT 1 R). Dose dependent increase in AT 1 R gene increases blood pressure in transgenic mice. Human AT 1 R gene promoter has four single nucleotide polymorphisms (SNPs) ( T/A at -777, T/G at -680, A/C at -214, and A/G at -119). Our studies have shown that variants -777T, -680T, -214A, and -119A almost always occur together in Caucasian subjects, creating haplotype I (TTAA) and II (AGCG) respectively. We have analyzed the genomic DNA from 388 normotensive and 374 hypertensive subjects and shown that haplotype I of the h AT 1 R gene is associated with hypertension in Caucasian subjects (p=&lt; 0.001, χ 2 = 12.26). Our transient transfection studies in adrenal cortical (H295R) and vascular smooth muscle (VSMC) cells show that haplotype I of h AT 1 R gene has increased basal and induced promoter activity as compared to haplotype II. Our gel shift assays have shown that transcription factor USF-2, C/EBP beta and delta bind strongly to an oligonucleotide containing nucleoside A at -214 (present in haplotype-I). In order to understand the physiological effect of these haplotypes in an in vivo situation, we have generated transgenic mice containing 160Kb long BAC DNA (containing 67Kb of the 5’-flanking region, all five exons and four introns, and 44 Kb of the 3’-UTR of hAT 1 R gene) and differ only in 1.2Kb promoter containing either haplotype I or haplotype II. Our studies show that 8 weeks old male transgenic mice containing haplotype I have increased h AT 1 R mRNA level in the kidney (5.5 fold) and heart (3.7 fold) as compared to haplotype II. We also show that transgenic mice containing haplotype-I have increased systolic blood pressure (7 mmHg) as compared to transgenic mice containing haplotype II. Our results suggest that increased expression of haplotype I of the hAT 1 R gene may be a contributing factor for hypertension in Caucasian subjects.

Differential Regulation of Blood Pressure in Transgenic Mice Containing ‐217A/G Polymorphism of Human Angiotensinogen Gene in Response to Immobilization Stress

Stress is a risk factor for hypertension. However, the mechanism by which stress leads to hypertension is unclear. Stress induces glucocorticoid secretion. Increased glucocorticoids trigger DNA binding activity of C/EBPβ and C/EBPδ via glucocorticoid receptors (GR). Here we show that expression of C/EBPβ, C/EBPδ and AGT genes are significantly increased in adrenal medulla, liver and kidney of rats subjected to Immobilization stress (IS).Renin‐angiotensin system (RAS) plays an important role in regulating blood pressure. Transcription factors C/EBPβ and C/EBPδ are important in regulation of human angiotensinogen (hAGT) gene. We have shown that hAGT gene contains an A/G polymorphism at ‐217, and frequency of ‐217A allele is increased in African‐American hypertensive subjects. Nucleotide sequence of hAGT gene promoter containing ‐217A has homology with the consensus CCAAT/enhancer binding protein (C/EBP) and GR binding site. Therefore, we have generated transgenic mice containing either ‐217A or ‐217G haplotype of the hAGT gene. These transgenic mice were subjected to IS. We found that mice containing haplotype ‐217A subjected to IS has 1.72 fold increased expression of hAGT in liver and 8mmHg increase in blood pressure. Whereas the mice containing haplotype ‐217G shows only 1.2 fold increase in hAGT expression and no significant change in blood pressure. These results suggest that stress leads to the differential regulation of human AGT gene.

Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

Genetics of Salt-Sensitive Hypertension

The assessment of salt sensitivity of blood pressure is difficult because of the lack of universal consensus on definition. Regardless of the variability in the definition of salt sensitivity, increased salt intake, independent of the actual level of blood pressure, is also a risk factor for cardiovascular morbidity and mortality and kidney disease. A modest reduction in salt intake results in an immediate decrease in blood pressure, with long-term beneficial consequences. However, some have suggested that dietary sodium restriction may not be beneficial to everyone. Thus, there is a need to distinguish salt-sensitive from salt-resistant individuals, but it has been difficult to do so with phenotypic studies. Therefore, there is a need to determine the genes that are involved in salt sensitivity. This review focuses on genes associated with salt sensitivity, with emphasis on the variants associated with salt sensitivity in humans that are not due to monogenic causes. Special emphasis is given to gene variants associated with salt sensitivity whose protein products interfere with cell function and increase blood pressure in transgenic mice.

Differential effect of two haplotypes of the human angiotensinogen gene on blood pressure in transgenic mice

Angiotensinogen (AGT) gene locus is associated with human essential hypertension and over‐expression of the AGT gene increases blood pressure in transgenic mice. Human AGT gene has A/G polymorphic sites at ‐6 and ‐217 positions in its promoter. The frequency of AA haplotype of the human AGT gene containing ‐6A:‐217A is significantly increased in African‐American hypertensive patients as compared to the AG haplotype‐6A:‐217G. Reporter constructs containing AA haplotype of the AGT gene promoter have increased promoter activity on transient transfection in human liver cells as compared to the AG haplotype. In order to understand the role of AA and AG haplotypes on hAGT expression in an in vivo situation, we have generated double transgenic mice containing either haplotype AA or AG of the hAGT gene (using knock‐in strategy at the HPRT locus) and human renin gene. Our studies show that transgenic mice containing AA haplotype have 1.6 fold increase in the AGT mRNA level in the liver as compared to the transgenic mice containing AG haplotype. In addition, Western blot and immunohistochemistry shows that liver and kidney of transgenic mice containing AA haplotype have increased hAGT protein level as compared to AG haplotype. In addition, blood pressure of three months old male transgenic mice containing AA haplotype is increased by about 8‐10 mmHg as compared to the transgenic mice containing AG haplotype. Supported by NHLBI.

A growing chain of evidence linking genetic variation in angiotensinogen with essential hypertension: focus on “A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G,” by Jain et al.

the prevalence of hypertension across the globe continues to rise despite advances in treatment and an ever-growing battery of antihypertensive drugs. Untreated hypertension remains an important risk factor for cardiovascular diseases, including stroke. The renin-angiotensin system (RAS) has long

A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

The human angiotensinogen (hAGT) gene contains an A/G polymorphism at -217, and frequency of -217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant -217A almost always occurs with -532T, -793A, and -1074T, whereas variant -217G almost always occurs with -532C, -793G, and -1074G. Since allele -6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, -6A:-217A (AA) and -6A:-217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by approximately 40%, and plasma angiotensin II level is increased by approximately 50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure.

Hepatic lipid accumulation in apolipoprotein C-I-deficient mice is potentiated by cholesteryl ester transfer protein

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.

Roles of Adrenomedullin in Hypertension and Hypertensive Organ Damage

Adrenomedullin (AM) is a bioactive peptide having a wide range of biological actions such as vasodilatation, natriuresis, diuresis and inhibition of aldosterone secretion. Various organs and tissues, including the myocardium, vascular wall and kidneys, produce AM, and AM is also present in the bloodstream. Plasma levels of AM were elevated in patients with essential or secondary hypertension as compared with normotensive controls. When infused in a relatively short period of time, AM reduced blood pressure in humans and experimental animals largely through vasodilatation. The blood pressure of transgenic mice overexpressing AM was lower than that of their wild-type littermates; while heterozygotes of AM knockout mice showed higher blood pressure, suggesting a role for endogenous AM in the regulation of blood pressure. Studies with cultured cardiac cells suggest a role for AM in inhibiting hypertrophy or fibrosis of the heart as an autocrine or paracrine factor. Animal experiment studies showed that either prolonged infusion or virally mediated overexpression of AM ameliorated the cardiovascular and renal damage associated with hypertension. Thus, a body of evidence accumulated in this field suggests that AM functions to counteract the elevation in blood pressure and progression of hypertensive organ damage.

Development of Hypertension and Kidney Hypertrophy in Transgenic Mice Overexpressing ARAP1 Gene in the Kidney

Angiotensin II regulates blood pressure via activation of the type 1 receptor. We previously identified a novel angiotensin II type 1 receptor-associated protein and demonstrated that it promotes receptor recycling to the plasma membrane. To delineate the pathophysiological function of the ARAP1 in the kidneys, we generated transgenic mice that overexpress rat ARAP1 cDNA specifically in proximal tubules and tested the hypothesis that proximal tubule-specific overexpression of ARAP1 causes hypertension. Two lines of male transgenic mice, 650 and 670, displayed kidney-specific transgene expression. Systolic blood pressure was significantly elevated by &20 to 25 mm Hg in these lines of mice at 20 weeks of age compared with their nontransgenic litter mates. Urine volume, but not water intake, was significantly decreased in both lines compared with nontransgenic controls. The kidney/body weight ratio was significantly increased in both lines compared with their nontransgenic litter mates at 12 and 20 weeks of age. In contrast, no difference was observed in the ratio of brain, spleen, heart, and testis to body weight between male transgenic and nontransgenic animals. Inhibitions of the renin-angiotensin system completely normalized the systolic blood pressure of transgenic mice. Moreover, low salt intake prevented the development of hypertension, whereas high salt intake exacerbated the increase in blood pressure in transgenic mice. Therefore, our data show that proximal tubule-specific overexpression of ARAP1 leads to hypertension, suggesting that renal ARAP1 plays an important role in the regulation of blood pressure and renal function via activation of the intrarenal renin-angiotensin system.

Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule.

The purpose of this study was to evaluate the physiological significance of a tissue renin-angiotensin system in the proximal tubule of the kidney. To accomplish this, we produced mice that express human renin (hREN) under the control of the kidney androgen-regulated promoter (KAP), which is androgen responsive. One of the lines expressed the hREN transgene primarily in the kidney. Renal expression of the transgene was undetectable in females but could be induced by testosterone treatment. Because the renin-angiotensin system is species specific, we bred KAP2-hREN mice with the mice expressing human angiotensinogen under the same promoter (KAP-hAGT) to produce offspring that expressed both transgenes. We measured mean arterial blood pressure (MAP) in the carotid artery of double-transgenic and control mice using radiotelemetry. Double-transgenic female mice had a normal baseline MAP (116 +/- 4 mmHg, n = 8), which increased by 15 mmHg after 2 wk of testosterone treatment, and returned to baseline after elimination of the testosterone pellet. The change in arterial pressure paralleled the change in plasma testosterone. There was no MAP change in testosterone-treated control littermates. We conclude that dual production of renin and angiotensinogen in the renal proximal tubule can result in a systemic increase in arterial pressure. These data support a role for a tissue-specific renin-angiotensin system in the renal proximal tubule that contributes to the regulation of systemic blood pressure.

Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence.

NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis. We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production. DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.

It’s Renin in the Brain

Since the first description of renin, angiotensinogen and angiotensin as well as converting enzyme and angiotensin receptors in the brain exactly 30 years ago,1–5⇓⇓⇓⇓ there was a heated debate, whether an endogenous intrinsic brain renin- angiotensin system (RAS) exists or not.6,7⇓ For a long time, the “believers” of local angiotensin II generation by the brain RAS were in a minority. Even at a symposium 1981, celebrating the 10-year anniversary of the discovery of the brain RAS, there was skepticism and the question whether the “renin-like” enzyme in brain was identical with cathepsins, tonin, or other proteases was a matter of debate. Historically, it is interesting that the presence in the brain of other classically peripheral peptides such as insulin, glucagon, and substance P was accepted without identification of the local synthetic pathway and less controversial than angiotensin as neuropeptide. Open letters, public debates, and personal accusations nourished sometimes emotional controversy whether angiotensin could be generated locally in tissue. The brain was a model for angiotensin generating pathways in other extrarenal tissues because the blood-brain barrier, which is impermeable for proteins and peptides, made it more unlikely that the RAS components measured in the brain were contaminations from the plasma. Angiotensin has extremely powerful effects on the brain that synergistically increase blood volume and blood pressure, eg, by stimulation of water intake and salt appetite, release of various pituitary hormones, increase of sympathetic tone, and decrease of baroreceptor reflex.7 The local generation of angiotensin II in the brain by an endogenous intrinsic RAS could therefore be extremely important for cardiovascular control and beyond. With the advent of transgenic technology, novel approaches to study the mechanistic and functional aspects of the brain RAS became available. Renin and angiotensinogen could be overexpressed or ablated …

Elevated blood pressure in transgenic mice with brain-specific expression of human angiotensinogen driven by the glial fibrillary acidic protein promoter.

In addition to the circulatory renin (REN)-angiotensin system (RAS), a tissue RAS having an important role in cardiovascular function also exists in the central nervous system. In the brain, angiotensinogen (AGT) is expressed in astrocytes and in some neurons important to cardiovascular control, but its functional role remains undefined. We generated a transgenic mouse encoding the human AGT (hAGT) gene under the control of the human glial fibrillary acidic protein (GFAP) promoter to experimentally dissect the role of brain versus systemically derived AGT. This promoter targets expression of transgene products to astrocytes, the most abundant cell type expressing AGT in brain. All transgenic lines exhibited hAGT mRNA expression in brain, with variable expression in other tissues. In one line examined in detail, transgene expression was high in brain and low in tissues outside the central nervous system, and the level of plasma hAGT was not elevated over baseline. In the brain, hAGT protein was mainly localized in astrocytes, but was present in neurons in the subfornical organ. Intracerebroventricular (ICV) injection of human REN (hREN) in conscious unrestrained mice elicited a pressor response, which was abolished by ICV preinjection of losartan. Double-transgenic mice expressing the hREN gene and the GFAP-hAGT transgene exhibited a 15-mm Hg increase in blood pressure and an increased preference for salt. Blood pressure in the hREN/GFAP-hAGT mice was lowered after ICV, but not intravenous losartan. These studies suggest that AGT synthesis in the brain has an important role in the regulation of blood pressure and electrolyte balance.

Vascular function and blood pressure in GH transgenic mice.

Acromegaly is associated with cardiovascular disease. We studied vascular function and mean arterial blood pressure in transgenic mice overexpressing bovine GH. Mean arterial blood pressure was measured in conscious, unrestrained male and female bovine GH and littermate control mice during normal as well as high salt intake using telemetric devices. Structure in artificially perfused maximally dilated hindquarter vascular beds and vascular reactivity and endothelial function in small mesenteric vessels were studied in female bovine GH and control mice. Mean arterial blood pressure was increased in female bovine GH transgenic (126 +/- 3 mm Hg) and male bovine GH transgenic (129 +/- 4 mm Hg) compared with female (109 +/- 3 mm Hg, P < 0.05) and male (111 +/- 3 mm Hg, P < 0.05) controls respectively. Increased salt intake had no effect on mean arterial blood pressure. Perfusion studies showed a significant decrease in the average diameter of the female bovine GH transgenic hindquarter vascular bed (P < 0.05). The responses of isolated resistance arteries to nor-epinephrine, potassium-induced depolarization, acetylcholine, or sodium-nitroprusside did not significantly differ between bovine GH transgenic and control mice. We conclude that the phenotype of the bovine GH transgenic mice includes a salt-resistant form of hypertension. Furthermore, the increase in mean arterial blood pressure is accompanied by a significant structural narrowing of the resistance vasculature without changes in vascular reactivity or endothelial function. The results imply that hypertension in bovine GH transgenic mice is maintained mainly by a structurally based increase in peripheral vascular resistance.

Hyperlipidaemia is associated with increased insulin-mediated glucose metabolism, reduced fatty acid metabolism and normal blood pressure in transgenic mice overexpressing human apolipoprotein C1.

Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL). Basal and hyperinsulinaemic (6 mU.kg-1.min-1), euglycaemic (7 mmol/l) clamps with 3(-)3H-glucose or 9,10(-)3H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out. The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 +/- 1.2 vs 1.6 +/- 0.1, 9.1 +/- 2.3 vs 0.6 +/- 0.1, 1.9 +/- 0.2 vs 0.9 +/- 0.1 and 7.0 +/- 1.0 vs 10.0 +/- 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 +/- 2 vs 10 +/- 1 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of 3H2O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 +/- 28 vs 200 +/- 11; 183 +/- 39 vs 128 +/- 17 and 156 +/- 44 vs 72 +/- 17 mumol.kg-1.min-1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-(3)H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 +/- 21 vs 314 +/- 88 ml.kg-1.min-1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of 3H2O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 +/- 3 vs 33 +/- 6; 3 +/- 2 vs 11 +/- 4 and 12 +/- 2 vs 22 +/- 4 mumol.kg-1.min-1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 +/- 0.3 vs 0.5 +/- 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 +/- 4 vs 85 +/- 3 mm Hg and 459 +/- 14 vs 484 +/- 11 beats.min-1). 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure.