Birgitta Kimura

Levels of angiotensin and molecular biology of the tissue renin angiotensin systems

The cloning of renin, angiotensinogen and angiotensin converting enzyme genes have established a widespread presence of these components of the renin-angiotensin system in multiple tissues. New sites of gene expression and peptide products in different tissues has provided strong evidence for the production of angiotensin independently of the endocrine blood borne system. In addition, the cloning of the angiotensin receptor (AT1) gene has confirmed the widespread distribution of angiotensin and suggested new functions for the peptide. This review of various tissues shows the variation in gene expression between tissues and angiotensin levels, and the fragmentary state of our knowledge in this area. As yet we cannot state that the gene expression of the substrates, enzymes and peptide products are involved in a single cell synthesis. This is not so much evidence against a paracrine function for tissue angiotensin, as lack of detailed, accurate intracellular information. The low abundance of renin in brain, spleen, lung and thymus compared to kidney, adrenal, heart, testes, and submandibular gland may suggest that there are both tissue renin-angiotensin systems (RAS) and nonrenin-angiotensin systems (NRAS). The NRAS could function through cleavage of angiotensinogen by serine proteinases such as tonin and cathepsin G to form Ang II directly. Although much angiotensinogen is extracellular and could therefore be a site of synthesis outside of the cell, intracellular angiotensinogen in a NRAS process could produce Ang II intracellularly without requiring extracellular conversion of Ang I to Ang II by ACE. In summary, renin mRNA is found in high concentrations in kidney, adrenal and testes and decreasing lower concentrations in ovary, liver, brain, spleen, lung and thymus. Angiotensinogen mRNA is found in the following tissues in descending order of abundance: liver, fat cells, brain (glial cells), kidney, ovary, adrenal gland, heart, lung, large intestine and stomach. It is debatable whether angiotensinogen and renin mRNA are expressed in blood vessels. The evidence that is lacking for a paracrine function of angiotensin is a complete description of the intracellular molecular synthesis and release of Ang II from single cells of promising tissues. Such tissues, SMG, ovary, testes, adrenal, pituitary and brain (neurons and glia) are potent sources of RAS components for future studies. Although the evidence for a paracrine function of angiotensin II is incomplete, it is an important concept for progressing toward the understanding of tissue peptide physiology and the significance of their gene regulation.

Prolonged Reduction of High Blood Pressure With an In Vivo, Nonpathogenic, Adeno-Associated Viral Vector Delivery of AT1-R mRNA Antisense

To produce a prolonged decrease in blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV does not stimulate inflammation or immune reaction. AAV enters nondividing cells and does not replicate. Therefore, it is an appropriate choice for gene therapy. Recombinant AAV was prepared with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1 receptor inserted in the antisense direction. The cassette was packaged in the virion. Stable transfection of NG108-15 cells with the PAAV-AS (plasmid AAV) antisense to AT1-R produced a significant reduction in AT1 receptors. A single injection of the rAAV-AS (viral vector) was made in adult spontaneously hypertensive rats, either directly in the hypothalamus (1 microL) or in the lateral ventricles (5 microL). The result shows that there is a significant decrease of blood pressure (approximately 23 +/- 2 mm Hg) for up to 9 weeks after injection. Control injections of mock vector produced no change in blood pressure during the same time period in age-matched controls. In young spontaneously hypertensive rats (3 weeks), a single intracardiac injection of recombinant rAAV-AS reduced blood pressure and slowed the development of hypertension compared with controls (P < .01). The results suggest that a prolonged reduction in high blood pressure can be achieved with AAV vectors delivering antisense to inhibit AT1 receptors with a single administration.

Changes in skin angiotensin II receptors in rats during wound healing

Angiotensin (AII) is associated with increased vascular smooth muscle growth and we have found increased levels of tissue AII during healing of wounded skin. Here we have determined changes in skin AII receptors during wound healing in adult male Sprague-Dawley rats. An abdominal surgical incision was made under anesthesia and rats were sacrificed at different times after wounding. Specific binding of 125I-AII was significantly decreased at 12, 18 and 24 hours in the wounded tissue compared to control tissue from the same rat. By 3 days the binding had recovered to baseline levels. Receptors were mostly AT1, with a high and a low affinity site in the skin both in control and healing tissue. The Bmax of the high affinity site was significantly decreased in healing tissue but there was no significant change in Kd. Our results demonstrate that adult rat skin contains predominantly AT1 receptors and also that these receptors are downregulated for 12-24 hours after wounding.

Ancient DNA from Nubian and Somali wild ass provides insights into donkey ancestry and domestication

Genetic data from extant donkeys (Equus asinus) have revealed two distinct mitochondrial DNA haplogroups, suggestive of two separate domestication events in northeast Africa about 5000 years ago. Without distinct phylogeographic structure in domestic donkey haplogroups and with little information on the genetic makeup of the ancestral African wild ass, however, it has been difficult to identify wild ancestors and geographical origins for the domestic mitochondrial clades. Our analysis of ancient archaeological and historic museum samples provides the first genetic information on the historic Nubian wild ass (Equus africanus africanus), Somali wild ass (Equus africanus somaliensis) and ancient donkey. The results demonstrate that the Nubian wild ass was an ancestor of the first donkey haplogroup. In contrast, the Somali wild ass has considerable mitochondrial divergence from the Nubian wild ass and domestic donkeys. These findings resolve the long-standing issue of the role of the Nubian wild ass in the domestication of the donkey, but raise new questions regarding the second ancestor for the donkey. Our results illustrate the complexity of animal domestication, and have conservation implications for critically endangered Nubian and Somali wild ass.

Reduction of Cold-Induced Hypertension by Antisense Oligodeoxynucleotides to Angiotensinogen mRNA and AT1-Receptor mRNA in Brain and Blood

Rats exposed chronically to mild cold (5 degrees C/41 degrees F) develop hypertension and cardiac hypertrophy. This provides a unique model of hypertension that is environmentally induced. The blood renin-angiotensin system (RAS) has been shown to play a role in both initiating and maintaining the high blood pressure (BP) in cold-induced hypertension. The mechanism also appears to involve both the tissue and brain RAS because there is increased mRNA for angiotensinogen (AGT) and angiotensin type 1 (AT1) receptors in brain and peripheral tissues, an increased spontaneous drinking response, and an increased dipsogenic response to acute administration of angiotensin II (Ang II) in cold-treated rats. Antisense oligodeoxynucleotides (AS-ODN), targeted to the RAS, have been shown to reduce BP in spontaneously hypertensive rats. Therefore, we injected AS-ODN in rats with cold-induced hypertension to test whether antisense inhibition was effective in reducing this nongenetic nonsurgical hypertension. Sprague-Dawley rats were made hypertensive by cold exposure and injected intracerebroventricularly with AS-ODN to AGT mRNA (n=6) or AT1 receptor mRNA (n=6). Systolic BP was recorded by tail cuff 24 hours later for 2 or 7 days, respectively. Systolic BP decreased significantly in response to AGT-AS-ODN (40+/-6 mm Hg, P<0.01) within 1 day after injection and to AT1 receptor-AS-ODN (P<0.05) for 3 days after injection. The maximum decrease was 41+/-10 mm Hg. Systolic BP then gradually increased to the preinjection level. The spontaneous drinking response to cold treatment also decreased significantly (P<0.05) after AGT-AS-ODN or AT1 receptor-AS-ODN intracerebroventricular injection. Intracardiac injection of AT1-AS-ODN (n=6) reduced systolic BP by 36+/-8 mm Hg (P<0.05) and decreased AT1 receptor as measured by autoradiography in aorta, adrenal glands, and kidneys 24 hours after injection. These data show that AS-ODN reduces BP in cold-induced hypertension and that the hypertension involves both peripheral tissues and central RAS in addition to blood-borne RAS mechanisms.

Losartan potassium, a nonpeptide antagonist of angiotensin II, chronically administered p.o. does not readily cross the blood-brain barrier

Recently several novel nonpeptide antagonists of angiotensin II (Ang II) have been identified. One of these, losartan potassium (formerly DuP 753) was developed as an orally active and highly selective antagonist for Ang II. As it is inhibited by sulfhydryl agents, it is specific for the AT1 receptor subtype. Since Ang II has both central and peripheral effects, we investigated whether losartan, given p.o. chronically, crosses the blood-brain barrier. The effects of chronic administration of losartan orally (p.o.) at 3 mg/kg per day for three days on the dipsogenic and pressor responses to a pre-established dose of Ang II i.v.t. (50 ng) were studied. Three series of experiments were carried out using conscious normotensive Sprague-Dawley rats. The rats were injected with Ang II intraventricularly (i.v.t.) before and after treatment of losartan p.o. and blood pressure and drinking responses measured. The experiments established that 3 mg/kg losartan p.o. for 3 days antagonized pressor effects of Ang II intravenously (i.v.), but did not antagonize the pressor or drinking effects of Ang II i.v.t. Daily water intake significantly increased with chronic losartan p.o.. Since chronic administration of losartan p.o. was able to block the effects of Ang II i.v. but had no effect on Ang II i.v.t. we conclude that losartan potassium does not readily cross the blood-brain barrier using this dose regimen.

Attenuation of Hypertension and Heart Hypertrophy by Adeno-Associated Virus Delivering Angiotensinogen Antisense

Angiotensinogen (AGT), one of the major components in the renin-angiotensin system, has been linked to hypertension in humans and animals. We have previously systemically administered antisense oligonucleotides and plasmid vectors with DNA that targeted AGT and attenuated hypertension in spontaneously hypertensive rats. The aim of the present study was to prolong the effect of antisense treatment by the use of a recombinant adeno-associated viral (rAAV) vector targeted to AGT. Using a model of lifelong hypertension in which 5-day-old spontaneously hypertensive rats are treated, a single intracardiac injection of rAAV-AGT-antisense (rAAV-AGT-AS) delayed the onset of hypertension for 91 days and significantly attenuated hypertension in adulthood for up to 6 months. Systolic blood pressure was always lower, by up to 23 mm Hg in the AS-treated group. The vector was stable and expressed a reporter gene in liver, kidney, and heart. The rAAV-AGT-AS treatment significantly decreased left ventricular hypertrophy (P =0.01) and also lowered levels of AGT in the liver (2.78±0.61 &mgr;g/g tissue versus 5.23±0.41 &mgr;g/g tissue for the sense-treated group, P <0.01). Measurement of liver transaminases showed no evidence for liver toxicity. We conclude that rAAV-AGT-AS offers a safe, stable approach for gene therapy of hypertension.

Atrial natriuretic peptide as a marker for doxorubicin‐induced cardiotoxic effects

Doxorubicin is an effective antineoplastic agent, but it frequently causes dose‐related cardiotoxic effects. Because the atrial natriuretic peptide (ANP) level is elevated in children with heart defects, the authors measured the ANP levels in children to determine whether ANP might serve as a simple diagnostic indicator of cardiotoxic effects. Sixteen patients, 5 to 19 years of age, who were being treated with doxorubicin (45 mg/m2 body surface area) for various malignancies had ANP levels measured in plasma. There was a group of six children, with a significant peak of plasma ANP (pANP) levels 3 weeks after the administration of the drug. Of these six patients, five had received high cumulative doses of doxorubicin (160 to 370 mg/m2), and two of them went into congestive heart failure without a previous decline in left ventricular ejection fraction, a standard technique for monitoring cardiac function during treatment with doxorubicin. The other ten patients had normal ANP levels throughout the study, and signs of cardiac dysfunction did not develop. None of the patients in the control group who had cancer and were not treated with doxorubicin and none of the healthy volunteers had elevated ANP levels. These preliminary results suggest that pANP may be useful as an early and sensitive indicator for doxorubicin‐related myocardial damage. Cancer 1992; 69:1492‐1497.

Critical Role of AT1 Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts : Beneficial Effect of Antisense Oligodeoxynucleotides Directed at AT1 Receptor mRNA

To examine the relevance of angiotensin II type 1 receptor (AT1R) expression in the determination of myocardial function after ischemia/reperfusion, Sprague-Dawley rats were treated intravenously with antisense oligodeoxynucleotides (AS-ODNs) directed at AT1R mRNA (100 microg/rat, n=9) or scrambled antisense oligodeoxynucleotides (Scr-ODNs, 100 microg/rat, n=6). Both AS-ODNs and Scr-ODNs were given along with 300 microg/rat of liposome DOTAP/DOPE, a positive electron carrier (wt:wt= 1:1). The hearts from AS-ODN- or Scr-ODN-treated rats were excised 24 hours later, perfused in vitro, and subjected to 25 minutes of global ischemia followed by 30 minutes of reperfusion. Parallel groups of rats were given the specific AT1R antagonist losartan (10 mg/kg IV, n=6) or saline (n=7) 4 to 6 hours before excising the hearts. Ischemia/reperfusion resulted in a significant increase in myocardial AT1R expression (autoradiography and binding assay) and myocardial dysfunction, indicated by increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in developed left ventricular pressure (all P<0.01 versus baseline) in the saline-treated group. AT1R protein and mRNA levels also increased in ischemic/ reperfused myocardial tissues. Administration of AS-ODNs or losartan, but not Scr-ODNs, preserved myocardial function and blocked the increased AT1R binding after ischemia/reperfusion (both P<0.01). Myocardial AT1R mRNA levels were not affected by either AS-ODNs or losartan, and the AT1R protein levels were significantly reduced by AS-ODN, but not losartan, treatment. Plasma angiotensin II levels increased after administration of losartan but not after administration of AS-ODNs. These observations imply a critical role of AT1R upregulation in determining myocardial function immediately after ischemia/reperfusion. AS-ODNs to AT1R mRNA may be more beneficial than losartan, because losartan does not affect the plasma angiotensin II level. The sustained increase in AT1R mRNA, but diminished protein expression, in rat hearts treated with AS-ODNs suggests that AS-ODNs block AT1R at the translational level.

Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices

Among the many functions of angiotensin II (Ang II) it now appears that Ang II is a growth factor. The concentration of Ang II in rat skin has been shown to increase during wound healing. To investigate the intracellular effect of Ang II in skin we determined the levels of total cytoplasmic inositol phosphates after incubation of skin slices with different doses of Ang II. 10(-6) M of Ang II increased significantly the phosphatidylinositol (PI) hydrolysis, and the effect was dose dependent up to 10(-4) M Ang II. The majority of inositol phosphates yielded after 1 hour incubation in the presence of lithium was InsP1, with lesser amount of InsP2. Losartan, the Ang II AT1 antagonist, at a dose of 10(-4) M blocked the effect of Ang II, while PD123319, the Ang II AT2 antagonist, had no antagonistic action; PD123319 at the higher dose of 10(-3) M, however, potentiated the effect of Ang II on PI hydrolysis. The results suggest that PI hydrolysis is a second messenger system for Ang II in rat skin. Also, the two subtypes of Ang II receptors mediate opposite effects on PI hydrolysis: Ang II binding to AT1 receptors increases inositol phosphate production, while Ang II binding to AT2 receptors decreases inositol phosphate production.