Yoshihiko Kakinuma

Renin-dependent cardiovascular functions and renin-independent blood-brain barrier functions revealed by renin-deficient mice.

Renin plays a key role in controlling blood pressure through its specific cleavage of angiotensinogen to generate angiotensin I (AI). Although possible existence of the other angiotensin forming enzymes has been discussed to date, its in vivo function remains to be elucidated. To address the contribution of renin, we generated renin knockout mice. Homozygous mutant mice show neither detectable levels of plasma renin activity nor plasma AI, lowered blood pressure 20–30 mm Hg less than normal, increased urine and drinking volume, and altered renal morphology as those observed in angiotensinogen-deficient mice. We recently found the decreased density in granular layer cells of hippocampus and the impaired blood-brain barrier function in angiotensinogen-deficient mice. Surprisingly, however, such brain phenotypes were not observed in renin-deficient mice. Our results demonstrate an indispensable role for renin in the circulating angiotensin generation and in the maintenance of blood pressure, but suggest a dispensable role for renin in the blood-brain barrier function.

IMPAIRED BLOOD-BRAIN BARRIER FUNCTION IN ANGIOTENSINOGEN-DEFICIENT MICE

Astrocytes in the central nervous system have physiologically important roles in the response to brain injury. Brain damage results in disruption of the blood–brain barrier (BBB), producing detachment of astrocyte endfeet from endothelial cells. The resultant leakage of serum proteins from loosened tight junctions between endothelial cells produces brain edema. At the same time, reactive astrocytes migrate to the injured area, where they proliferate and produce extracellular matrix, thereby reconstituting the BBB. As astrocytes are known to express angiotensinogen, which is the precursor of angiotensins (AI to AIV), we have investigated a possible functional contribution of angiotensinogen or one of its metabolites to BBB reconstitution. The astrocytes of angiotensinogen knockout mice had very attenuated expression of glial fibrially acidic protein and decreased laminin production in response to cold injury, and ultimately incomplete reconstitution of impaired BBB function. Although these abnormalities were rescued by administration of AII or AIV, the restoration of BBB function was not inhibited by AII type 1 and 2 receptor antagonists. These findings provide evidence that astrocytes with angiotensins are required for functional maintenance of the BBB.

Vascular Endothelin-B Receptor System In Vivo Plays a Favorable Inhibitory Role in Vascular Remodeling After Injury Revealed by Endothelin-B Receptor–Knockout Mice

Background—Two subtypes of endothelin (ET) receptors, ETA and ETB, are distributed in vascular smooth muscle cells to cause contraction and proliferation. Vascular endothelial cells express only ETB receptors, which cause NO release. Although ETA receptor blockade is reported to be effective in ameliorating vascular remodeling, there is no report on the long-term effect of ETB receptor blockade on vascular remodeling after injury. Methods and Results—ETB receptor–knockout (KO) mice, which were genetically rescued from lethal intestinal aganglionosis, and wild-type (WT) mice underwent complete ligation of the right common carotid artery, ie, a blood flow cessation model of vascular remodeling. Fourteen days after ligation, the intimal area, the ratio of intimal to medial areas, and the stenotic ratio in the ligated artery of KO mice were significantly increased compared with those of WT mice. The expression level of ET-1 mRNA in the ligated artery of KO mice was increased similarly to that of WT mice, whereas tissue NOx levels in lesions of KO mice were significantly lower than those of WT mice. Long-term treatment with the ETA receptor antagonist TA-0201 (0.5 mg · kg−1 · d−1) significantly ameliorated vascular stenosis in both groups. Long-term treatment with the ETB receptor antagonist A-192621 (30 mg · kg−1 · d−1) worsened vascular remodeling in WT mice. Conclusions—We demonstrated that inhibition of the ETB receptor system is harmful for vascular remodeling after injury, the mechanism of which is partly attributed to decreased NO release, in KO mice. These results suggest that the overall effect of vascular ETB receptors is antiproliferative in the injured artery.

Novel molecular mechanism of increased myocardial endothelin-1 expression in the failing heart involving the transcriptional factor hypoxia-inducible factor-1α induced for impaired myocardial energy metabolism

Background—Hypoxia-inducible factor (HIF)-1&agr; is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired &bgr;-oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1&agr; binding site in the 5′-promoter region of the ET-1 gene, we hypothesized that HIF-1&agr; is involved in this mechanism. Methods and Results—In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1&agr; transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5′-promoter region. HIF-1&agr; mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1&agr; mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1&agr; largely inhibited the increased gene expression of ET-1. Conclusions—The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1&agr; to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

Anti-apoptotic action of angiotensin fragments to neuronal cells from angiotensinogen knock-out mice

The morphological analysis in a congenic line of angiotensinogen knock-out mice (AgKO) revealed the decreased density in granular layer cells of hippocampus and cerebellum, suggesting neuronal cells of AgKO susceptible to apoptotic cell death. This phenomenon was further studied by culture of the hippocampal neurons with decreased concentration of serum. AgKO neuronal cells, which showed apoptosis by lower concentration of the serum within several hours, however, survived much longer in the presence of angiotensin II (AII) and IV (AIV). This anti-apoptotic action was not interfered by AII receptor antagonists, CV11874 and PD123319. These results suggest that the renin-angiotensin system could play a critical role in central nervous system, preventing neuronal cells from apoptosis not only by AII but also AIV.

Enhancement of glycolysis in cardiomyocytes elevates endothelin-1 expression through the transcriptional factor hypoxia-inducible factor-1 alpha.

We investigated whether the type of energy metabolism directly affects cardiac gene expression. During development, the heart switches from glycolysis to fatty acid beta-oxidation in vivo, as demonstrated by the developmental switching of the major isoform of myosin heavy chain (MHC) from beta to alpha. However, the beta-MHC isoform predominates in monocrotaline-induced pulmonary hypertension, a model of right ventricular hypertrophy in vivo. Cultured cardiomyocytes showed a predominance of beta-MHC expression over that of alpha-MHC, the same pattern as in the hypertrophied heart, suggesting that the in vitro condition itself causes the energy metabolism of cardiomyocytes to be switched to glycolysis. Electrical stimulation of cultured cardiomyocytes decreased the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxia-inducible factor-1 alpha (HIF-1 alpha), but not that of peroxisome-proliferator-activated receptor-gamma co-activator, suggesting that electrical stimulation suppresses the glycolytic system. Furthermore, a higher oxygen content (50%) decreased drastically the expression of GAPDH, HIF-1 alpha and endothelin-1 (ET-1), and increased [(3)H]palmitate uptake. These findings indicate that the intrinsic energy metabolic system in cultured cardiomyocytes in vitro is predominantly glycolysis, and that the gene expression of cardiac ET-1 parallels the state of the glycolytic system. An antisense oligonucleotide against HIF-1 alpha greatly decreased the gene expression of ET-1 and GAPDH, suggesting that cardiac ET-1 gene expression is regulated by cardiac energy metabolism through HIF-1 alpha. In conclusion, it is suggested that the pattern of gene expression of ET-1 reflects the level of the glycolytic system in cardiomyocytes, and that enhanced glycolysis regulates the cardiac gene expression of ET-1 via HIF-1 alpha.

Myocardial expression of endothelin-2 is altered reciprocally to that of endothelin-1 during ischemia of cardiomyocytes in vitro and during heart failure in vivo

We and other groups have reported that endothelin (ET)-1 expression in the heart is altered in the setting of heart diseases. We have also reported that myocardial ET-1 is involved in the progression of heart failure, and that an ET receptor antagonist improves long-term survival in heart failure (Nature 384: 353-355, 1996). However, the role of myocardial ET-2 in disease states are not known. To characterize the role of ET-2, we used a) the failing hearts of rats with heart failure caused by myocardial infarction, and b) primary cultured cardiomyocytes subjected to hypoxia. In the failing heart in vivo, ET-1 mRNA increased by 390% compared with that in the non-failing heart, while ET-2 mRNA drastically decreased by 88%. Thus, gene expression of ET-1 and ET-2 was reciprocally altered in the failing heart in vivo. In in vitro studies, reciprocal alterations in ET-1 and ET-2 gene expression were also observed in isolated primary cultured cardiomyocytes, subjected to hypoxia. Specifically, acute hypoxic stress induced a significant increase (360% of the basal level) in ET-2 mRNA expression compared with that in normoxic cells, whereas it decreased ET-1 mRNA expression by 62% in primary cultured cardiomyocytes. Although these two crucial conditions, i.e., heart failure in vivo and acute hypoxic stress in vitro, are pathophysiologically distinct from each other, reciprocal alteration of ET-1 and ET-2 gene expression was observed in both cases. To further investigate the regulatory mechanism of the altered gene expression, luciferase analysis was performed using primary cultured cardiomyocytes. ET-2 promoter, which is the 5-flanking region of preproET-2 gene (5ET-2), showed a marked increase in luciferase activity during acute hypoxia. In contrast, the luciferase activity of 5ET-1 (ET-1 promoter) did not change in response to hypoxic stress. The present study suggests that there are transcriptionally distinct regulatory mechanisms for ET-1 and ET-2 expression in cardiomyocytes, and therefore this study may provide a new aspect of cardiac ET system that not only ET-1 but also ET-2 can be participated in the pathophysiological conditions.

Endothelin-1 stimulates cardiomyocyte injury during mitochondrial dysfunction in culture

To understand the pathophysiological role of endothelin-1 in the failing heart, we constructed a cellular mitochondrial impairment model and demonstrated the effect of endothelin-1. Primary cultured cardiomyocytes from neonatal rats were pretreated with rotenone, a mitochondrial complex I inhibitor, and the cytotoxic effect of endothelin-1 on the cardiomyocytes was demonstrated. Rotenone gradually decreased the pH of the culture medium with incubation time and caused slight cell injury. Endothelin-1 markedly enhanced the effect of rotenone that decreased the pH of the medium and enhanced cellular injury. The enhancement of the decrease in pH and cell injury induced by endothelin-1 was counteracted by the endothelin ET(A) receptor antagonist BQ123 or by maintaining the pH of the medium by the addition of 50 mM HEPES. Endothelin-1 markedly increased the uptake of 2-deoxyglucose and lactic acid production when the cardiomyocytes were pretreated with rotenone. These findings suggest that the stimulation of glucose uptake and anaerobic glycolysis followed by the increase in lactic acid accumulation in cardiomyocytes under the condition of mitochondrial impairment may be involved, at least in part, in the cellular injury by endothelin-1. Moreover, these findings suggest the possibility that the effect of endothelin-1 on myocardium is reversed by the condition of the mitochondria, and endogenous endothelin-1 may deteriorate cardiac failure with mitochondrial dysfunction. This may contribute to clarify the beneficial effect of endothelin receptor blockade in improving heart failures.

Developmental stage-specific involvement of angiotensin in murine nephrogenesis

Abstractu2002Angiotensinogen-deleted mice (Agt-KO) show phenotypes of hypotension and renal atrophy. To investigate whether an alternative pathway other than angiotensin II (AII), i.e., processed angiotensin fragments, may play a biological role in nephrogenesis, we analyzed a congenic line of Agt-KO fetuses and neonates derived from two sources: one (Agt-KO/He) from mating with heterozygous angiotensinogen-deleted mice and the other (Agt-KO/Ho) from mating homozygous angiotensinogen-deleted mice. Although Agt-KO/He did not show a typical phenotype at birth, these mice showed papillary atrophy 2 weeks later and thereafter, a marked increase in renal size, i.e., pelvic dilatation. In contrast, Agt-KO/Ho showed renal abnormalities at birth and subsequently died. TUNEL staining and electron microscopy revealed that accelerated papillary apoptosis was present at birth in Agt-KO/Ho and caused abnormal papillary development; however, apoptosis was not detected in Agt-KO/He, suggesting that different mechanisms for the abnormal renal development exist in Agt-KO/He and Agt-KO/Ho. Two-week administration of an angiotensin fragment (3–8), angiotensin IV (AIV), to Agt-KO/He markedly attenuated the renal atrophy, decreasing the incidence from 81% to 14%. However, administration of AIV to fetal Agt-KO/Ho through the mother did not decrease the incidence. This is marked contrast to AII, which prevented renal atrophy in both fetal and neonatal periods. It is therefore suggested that AIV is involved in nephrogenesis in a developmental stage-specific manner.

Continuous intravenous administration of a low dose of epoprostenol greatly decreased serum concentrations of endothelin-1 in primary pulmonary hypertension--a case report.

Endothelin-1 (ET-1) is known to be a principal factor in the pathogenesis of primary pulmonary hypertension (PPH). Recently intravenous administration of epoprosterol improved the survival rate in PPH. However, the effect of epoprosterol on ET-1 remains to be investigated. Therefore, we studied a patient with PPH who was treated with a low dose of epoprosterol and examined the serum concentration of ET-1 during the treatment. Epoprosterol greatly decreased the serum concentration of ET-1 in parallel with improvement of the clinical course, suggesting that ET-1 level may be a marker for treatment of PPH.