Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy

Background

Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane—extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage—has been evaluated for its anticarcinogenic and antioxidant effects.

Aims

To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro.

Methods

Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways.

Results

We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB.

Conclusion

Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.     

Role of inflammation in the development of renal damage and dysfunction in angiotensin II-induced hypertension

Angiotensin II (Ang II)-induced hypertension is associated with an inflammatory response that may contribute to the development of target organ damage. We tested the hypothesis that, in Ang II-induced hypertension, CC chemokine receptor 2 (CCR2) activation plays an important role in the development of renal fibrosis, damage, and dysfunction by causing oxidative stress, macrophage infiltration, and cell proliferation. To test this hypothesis, we used CCR2 knockout mice (CCR2-/-). The natural ligand of CCR2 is monocyte chemoattractant protein-1, a chemokine important for macrophage recruitment and activation. CCR2-/- and age-matched wild-type (CCR2+/+) C57BL/6J mice were infused continuously with either Ang II (5.2 ng/10 g per minute) or vehicle via osmotic minipumps for 2 or 4 weeks. Ang II infusion caused similar increases in systolic blood pressure and left ventricular hypertrophy in both strains of mice. However, in CCR2-/- mice with Ang II-induced hypertension, oxidative stress, macrophage infiltration, albuminuria, and renal damage were significantly decreased, and glomerular filtration rate was significantly higher than in CCR2+/+ mice. We concluded that, in Ang II-induced hypertension, CCR2 activation plays an important role in the development of hypertensive nephropathy via increased oxidative stress and inflammation.     

Pancreatic polypeptide-fold peptide receptors and angiotensin II-induced renal vasoconstriction

The Gi pathway augments renal vasoconstriction induced by angiotensin II in spontaneously hypertensive but not normotensive Wistar-Kyoto rats. Because the Gi-coupled pancreatic polypeptide (PP)-fold peptide receptors Y1 and Y2 are expressed in kidneys and are activated by endogenous PP-fold peptides, we tested the hypothesis that these receptors regulate angiotensin II-induced renal vasoconstriction in kidneys from hypertensive but not normotensive rats. A selective Y1-receptor agonist [(Leu31,Pro34)-neuropeptide Y; 6 to 10 nmol/L] greatly potentiated angiotensin II-induced changes in perfusion pressure in isolated, perfused kidneys from hypertensive but not normotensive rats. A selective Y2-receptor agonist (peptide YY(3-36); 6 nM) only slightly potentiated angiotensin II-induced renal vasoconstriction and only in kidneys from hypertensive rats. Neither the Y1-receptor nor the Y2-receptor agonist increased basal perfusion pressure. BIBP3226 (1 micromol/L, highly selective Y1-receptor antagonist) and BIIE0246 (1 micromol/L, highly selective Y2-receptor antagonist) completely abolished potentiation by (Leu31,Pro34)-neuropeptide Y and peptide YY(3-36), respectively. Y1-receptor and Y2-receptor mRNA and protein levels were expressed in renal microvessels and whole kidneys, but the abundance was similar in kidneys from hypertensive and normotensive rats. Both Y1-receptor-induced and Y2-receptor-induced potentiation of angiotensin II-mediated renal vasoconstriction was completely abolished by pretreatment with pertussis toxin (30 microg/kg IV, blocks Gi proteins). These data indicate that, in kidneys from genetically hypertensive but not normotensive rats, Y1-receptor activation markedly enhances angiotensin II-mediated renal vasoconstriction by a mechanism involving Gi. Although Y2 receptors can also potentiate angiotensin II-mediated renal vasoconstriction via Gi, the effect is modest compared with Y1 receptors. These findings may have important implications for the etiology of genetic hypertension.     

Role of pressure in angiotensin II-induced renal injury: chronic servo-control of renal perfusion pressure in rats

Renal perfusion pressure was servo-controlled chronically in rats to quantify the relative contribution of elevated arterial pressure versus angiotensin II (Ang II) on the induction of renal injury in Ang II-induced hypertension. Sprague-Dawley rats fed a 4% salt diet were administered Ang II for 14 days (25 ng/kg per minute IV; saline only for sham rats), and the renal perfusion pressure to the left kidney was continuously servo-controlled to maintain a normal pressure in that kidney throughout the period of hypertension. An aortic occluder was implanted around the aorta between the two renal arteries and carotid and femoral arterial pressure were measured continuously throughout the experiment to determine uncontrolled and controlled renal perfusion pressure, respectively. Renal perfusion pressure of uncontrolled, controlled, and sham kidneys over the period of Ang II or saline infusion averaged 152.6+/-7.0, 117.4+/-3.5, and 110.7+/-2.2 mm Hg, respectively. The high-pressure uncontrolled kidneys exhibited tubular necrosis and interstitial fibrosis, especially prominent in the outer medullary region. Regional glomerular sclerosis and interlobular artery injury were also pronounced. Controlled kidneys were significantly protected from interlobular artery injury, juxtamedullary glomeruli injury, tubular necrosis, and interstitial fibrosis as determined by comparing the level of injury. Glomerular injury was not prevented in the outer cortex. Transforming growth factor (TGF)-beta and active NF-kappaB proteins determined by immunohistochemistry were colocalized in the uncontrolled kidney in regions of interstitial fibrosis. We conclude that the preferential juxtamedullary injury found in Ang II hypertension is largely induced by pressure and is probably mediated through the TGF-beta and NF-kappaB pathway.     

Interleukin 6 underlies angiotensin II-induced hypertension and chronic renal damage

Chronic kidney disease (CKD) is a prevalent life-threatening disease frequently associated with hypertension, progression to renal fibrosis, and eventual renal failure. Although the pathogenesis of CKD remains largely unknown, an increased inflammatory response is known to be associated with the disease and has long been speculated to contribute to disease development. However, the causative factors, the exact role of the increased inflammatory cascade in CKD, and the underlying mechanisms for its progression remain unidentified. Here we report that interleukin 6 (IL-6) expression levels were significantly increased in the kidneys collected from CKD patients and further elevated in CKD patients characterized with hypertension. Functionally, we determined that angiotensin II is a causative factor responsible for IL-6 induction in the mouse kidney and that genetic deletion of IL-6 significantly reduced hypertension and key features of CKD, including renal injury and progression to renal fibrosis in angiotensin II-infused mice. Mechanistically, we provide both human and mouse evidence that IL-6 is a key cytokine functioning downstream of angiotensin II signaling to directly induce fibrotic gene expression and preproendothelin 1 mRNA expression in the kidney. Overall, both the mouse and human studies reported here provide evidence that angiotensin II induces IL-6 production in the kidney, and that, in addition to its role in hypertension, increased IL-6 may play an important pathogenic role in CKD by inducing fibrotic gene expression and ET-1 gene expression. These findings immediately suggest that the IL-6 signaling is a novel therapeutic target to manage this devastating disorder affecting millions worldwide.     

Genetic deletion of the p66Shc adaptor protein protects from angiotensin II-induced myocardial damage

Angiotensin II (Ang II), acting through its G protein-coupled AT1 receptor (AT1), contributes to the precocious heart senescence typical of patients with hypertension, atherosclerosis, and diabetes. AT1 was suggested to transactivate an intracellular signaling controlled by growth factors and their tyrosin-kinase receptors. In cultured vascular smooth muscle cells, this downstream mechanism comprises the p66Shc adaptor protein, previously recognized to play a role in vascular cell senescence and death. The aim of the present study was 2-fold: (1) to characterize the cardiovascular phenotype of p66Shc knockout mice (p66Shc(-/-)), and (2) to test the novel hypothesis that disrupting the p66Shc might protect the heart from the damaging action of elevated Ang II levels. Compared with wild-type littermates (p66Shc(+/+)), p66Shc(-/-) showed similar blood pressure, heart rate, and left ventricular wall thickness. However, cardiomyocyte number was increased in mutant animals, indicating a condition of myocardial hyperplasia. In p66Shc(+/+), infusion of a sub-pressor dose of Ang II (300 nmol/kg body weight [BW] daily for 28 days) caused left ventricular hypertrophy and apoptotic death of cardiomyocytes and endothelial cells. In contrast, p66Shc(-/-) were resistant to the proapoptotic/hypertrophic action of Ang II. Consistently, in vitro experiments showed that Ang II causes apoptotic death of cardiomyocytes isolated from p66Shc(+/+) hearts to a greater extent as compared with p66Shc(-/-) cardiomyocytes. Our results indicate a fundamental role of p66Shc in Ang II-mediated myocardial remodeling. In perspective, p66Shc inhibition may be envisioned as a novel way to prevent the deleterious effects of Ang II on the heart.     

T regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury

Angiotensin (Ang) II induces hypertension by mechanisms mediated in part by adaptive immunity and T effector lymphocytes. T regulatory lymphocytes (Tregs) suppress T effector lymphocytes. We questioned whether Treg adoptive transfer would blunt Ang II-induced hypertension and vascular injury. Ten- to 12-week-old male C57BL/6 mice were injected IV with 3 ×10(5) Treg (CD4(+)CD25(+)) or T effector (CD4(+)CD25(-)) cells, 3 times at 2-week intervals, and then infused or not with Ang II (1 μg/kg per minute, SC) for 14 days. Ang II increased systolic blood pressure by 43 mm Hg (P<0.05), NADPH oxidase activity 1.5-fold in aorta and 1.8-fold in the heart (P<0.05), impaired acetylcholine vasodilatory responses by 70% compared with control (P<0.05), and increased vascular stiffness (P<0.001), mesenteric artery vascular cell adhesion molecule expression (2-fold; P<0.05), and aortic macrophage and T-cell infiltration (P<0.001). All of the above were prevented by Treg but not T effector adoptive transfer. Ang II caused a 43% decrease in Foxp3(+) cells in the renal cortex, whereas Treg adoptive transfer increased Foxp3(+) cells 2-fold compared with control. Thus, Tregs suppress Ang II-mediated vascular injury in part through anti-inflammatory actions. Immune mechanisms modulate Ang II-induced blood pressure elevation, vascular oxidative stress, inflammation, and endothelial dysfunction.     

Evidence for renal vascular remodeling in angiotensin II-induced hypertension

OBJECTIVES: To determine whether 'slow pressor' hypertension from systemic angiotensin (Ang II) infusion was associated with renal vascular structural remodeling of the renal resistance vessels and glomerulus. METHODS: Ang II (4.5-10 ng/kg per min) or vehicle was infused for 10 days. Renal resistance vascular lumen changes were assessed at 10 days as changes in renal pressure flow and pressure-glomerular filtration rate (GFR) and pressure-Na+ excretion in maximally dilated, isotonically perfused kidneys. RESULTS: Low-dose, initially subpressor Ang II infusion for 10 days increased conscious arterial pressure by 27 mmHg compared to vehicle-infused rats (140 +/- 7 and 113 +/- 2 mmHg, respectively). There was no change in the pressure-flow relationship but the slope of the pressure-GFR relationship was reduced in the rats treated with Ang II. These changes are consistent with equal and opposite pre-and post-glomerular effects (i.e., increased pre-glomerular vessel resistance and reduced post-glomerular vessel resistance) and reduced glomerular ultrafiltration coefficient. There was also a significant reduction in pressure-dependent Na+ excretion. CONCLUSIONS: Slow pressor Ang II-induced hypertension was associated with apparent pro-hypertensive changes in the kidney involving pre/post-glomerular vessel remodeling as indicated by an apparent reduction in pre-glomerular lumen dimensions, a reduced glomerular filtration capacity and a reduction in the pressure natriuresis relationship.     

Exogenous L-arginine ameliorates angiotensin II-induced hypertension and renal damage in rats

Experiments were performed to determine whether exogenous L-arginine could ameliorate angiotensin II-induced hypertension and renal damage. Rats were instrumented with chronic indwelling femoral venous and arterial catheters for infusions of drugs and measurement of conscious arterial pressure. Arterial blood pressure significantly increased from 124+/-1 to 199+/-4 mm Hg, after 9 days of continuous infusion of angiotensin II (20 ng/kg per minute; IV; n=6 to 9). In contrast, the increase in arterial pressure after 9 days of angiotensin II infusion was significantly blunted by 45% (P=0.0003) in rats coadministered L-arginine (300 microg/kg per minute; IV; n=7 to 9). The glomerular injury index was significantly greater in rats administered angiotensin II in comparison with rats administered saline vehicle (P<0.001). Coinfusion of L-arginine significantly increased plasma nitrate/nitrite concentrations (P<0.001) and completely prevented angiotensin II-induced glomerular damage (P<0.001). Angiotensin II infusion alone and combined angiotensin II plus L-arginine infusion significantly increased urinary albumin excretion. Albuminuria in rats administered angiotensin II plus L-arginine is likely to be because of increased intraglomerular pressure. Our experiments demonstrate that L-arginine can blunt angiotensin II-induced hypertension and associated renal damage. This latter observation is most exciting because it indicates that increasing NO bioavailability, in addition to lowering arterial pressure, can greatly reduce hypertension-induced renal damage.     

P450-dependent arachidonic acid metabolism and angiotensin II-induced renal damage

Transgenic rats overexpressing both human renin and angiotensinogen genes (dTGR) develop hypertension, inflammation, and renal failure. We tested the hypothesis that these pathological features are associated with changes in renal P450-dependent arachidonic acid (AA) metabolism. Samples were prepared from 5- and 7-week-old dTGR and from normotensive Sprague-Dawley (SD) rats, ie, before and after the dTGR developed severe hypertension and albuminuria. At both stages, dTGR showed significantly lower renal microsomal AA epoxygenase and hydroxylase activities that reached 63% and 76% of the control values at week 7. Furthermore, the protein levels of several potential AA epoxygenases (CYP2C11, CYP2C23, and CYP2J) were significantly reduced. Immunoinhibition studies identified CYP2C23 as the major AA epoxygenase, both in dTGR and SD rats. Immunohistochemistry showed that CYP2C23 was localized in cortical and outer medullary tubules that progressively lost this enzyme from week 5 to week 7 in dTGR. CYP2C11 expression occurred only in the outer medullary tubules and was markedly reduced in dTGR compared with age-matched SD rats. These findings indicate site-specific decreases in the availability of AA epoxygenase products in the kidney of dTGR. In contrast to renal microsomes, liver microsomes of dTGR and SD rats showed no change in the expression and activity of AA epoxygenases and hydroxylases. We conclude that hypertension and end-organ damage in dTGR is associated with kidney-specific downregulation of P450-dependent AA metabolism. Because the products of AA epoxygenation have anti-inflammatory properties, this alteration may contribute to uncontrolled renal inflammation, which is a major cause of renal damage in dTGR.     

In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3x10(10) plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.     

Levels of renal and extrarenal sympathetic drive in angiotensin II-induced hypertension

We examined the contribution of the renal nerves to mean arterial pressure (MAP) during 5-week chronic infusion of angiotensin II (Ang II; 50 ng/kg per minute SC) in conscious rabbits. Basal MAP was 68+/-1 mm Hg, and the maximum depressor response to ganglion blockade was -20+/-2 mm Hg. MAP increased by 25+/-2 mm Hg after 1 week and remained stable over the next 4 weeks. Depressor responses to pentolinium (6 mg/kg IV) were similar to control during the first week of hypertension but thereafter became increasingly greater in Ang II-treated rabbits but not vehicle-treated rabbits. After 5 weeks, the fall in MAP was 54% greater in Ang II- than in vehicle-treated rabbits (-34+/-2 versus -22+/-2 mm Hg), but renal sympathetic nerve activity was similar in both groups. Renal denervation produced a small fall in MAP in all of the vehicle-treated rabbits after 4 days (-6+/-2 mm Hg; P=0.01), but there was no consistent effect in hypertensive rabbits. The depressor response to ganglion blockade was enhanced in vehicle-treated but not Ang II-treated rabbits. The finding that renal sympathetic nerve activity is not altered by Ang II hypertension nor is the hypertension altered by renal denervation suggests that renal sympathetic nerves do not contribute to the hypertension. The greater depressor effect of acute ganglion blockade in hypertensive rabbits suggests that the sympathetic nervous system exerts increased vasoconstriction in the peripheral vasculature in Ang II-induced hypertension.     

Preferential inhibitory effect of nifedipine on angiotensin II-induced renal vasoconstriction

The inhibitory effects of nifedipine on renal vasoconstrictor response to angiotensin II, norepinephrine, or renal nerve stimulation were tested in anesthetized dogs. Intrarenal infusions of nifedipine (0.3, 1, and 3 micrograms/min) dose-dependently suppressed the renal vasoconstriction induced by intrarenal injections of angiotension II (0.03, 0.05, and 0.1 microgram) or norepinephrine (0.3-1 micrograms) but not that by renal nerve stimulation (4-7 Hz). However, the inhibitory effect of nifedipine on angiotensin II-induced vasoconstriction was greater than its effect on norepinephrine-induced or renal nerve stimulation-induced vasoconstriction (i.e., 50% reduction in renal blood flow). Furthermore, a greater renal vasodilation induced by intrarenal bolus injections of nifedipine (1,3, and 10 micrograms) but not by acetylcholine (0.1 and 0.3 microgram) was observed during the reduction in the perfusion pressure of the contralateral kidney to approximately 50 mm Hg, which resulted in an increase in plasma renin activity and plasma angiotensin II concentration but no change in plasma norepinephrine concentration. There was a significant positive correlation between plasma renin activity and plasma angiotensin II concentration before nifedipine injections and the subsequent increase in renal blood flow produced by each dose of nifedipine. These results indicate that nifedipine has a relatively preferential inhibitory effect on the renal vasoconstriction produced by both exogenous and endogenous angiotensin II in canine renal vasculature.     

Role of Rho-kinase and p27 in angiotensin II-induced vascular injury

Angiotensin II enhances the development of atherosclerotic lesion in which cellular proliferation and/or migration are critical steps. Although cyclin-dependent kinase inhibitor, p27, and Rho/Rho-kinase pathway have recently been implicated as factors regulating these events cooperatively, their role in vivo has not been fully elucidated. We evaluated the contribution of p27 and Rho-kinase to angiotensin II-induced vascular injury using p27-deficient mice. Two-week angiotensin II (1500 ng/kg per minute SC) infusion elicited similar degrees of elevation in systolic blood pressure in wild-type mice (159+/-5 mm Hg) and p27-deficient mice (157+/-5 mm Hg; P>0.05). Angiotensin II infusion to wild-type mice resulted in increases in the medial thickness of aorta, proliferating cell number, and monocyte/macrophage infiltration within the vasculature. In p27-deficient mice, however, these changes were more prominent than those in wild-type mice. Treatment of wild-type mice with fasudil, a selective Rho-kinase inhibitor, did not alter blood pressure but significantly upregulated p27 expression, decreased medial thickness of aorta, reduced proliferating cell number, and prevented monocyte/macrophage infiltration. These protective effects of fasudil were attenuated in p27-deficient mice. In conclusion, p27 constitutes an important modulator of angiotensin II-induced monocyte/macrophage infiltration and vascular remodeling, which is mediated in part by Rho-kinase stimulation. Inhibition of Rho-kinase activity improves angiotensin II-induced vascular injury through p27-dependent and p27-independent mechanisms.