Statins and angiotensin II-induced vascular injury

Ralf Dechend, Dominik Müller, Jeun Koon Park, Anette Fiebeler, Hermann Haller and Friedrich C. Luft

HELIOS Klinikum Berlin, Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Medical Faculty of the Charité, Humboldt University of Berlin, Germany and the Department of Nephrology, Hannover Medical School, Hannover, Germany

Effects of statins unrelated to lipid lowering

Statins may have pleiotropic properties that complement their cholesterol-lowering effects. These properties include nitric oxide-mediated improvement of endothelial dysfunction and attenuation of endothelin-1 expression, antioxidant effects, anti-inflammatory properties, inhibition of cell proliferation with anti-carcinogenic actions in animals, stabilization of atherosclerotic plaques, anti-coagulant effects and inhibition of graft rejection after heart and kidney transplantation [1]. In a remarkable short-term human study, Tsunekawa et al. [2] showed that cerivastatin improved endothelial dysfunction in elderly diabetic men within 3 days, independent of lipid lowering. The effect may have been partly due to upregulation of nitric oxide (NO) production.

Angiotensin II-induced vascular injury—a model to study non-clinical effects of statins

We have been interested in angiotensin II (Ang II)-induced vascular injury and were curious to see if a statin would ameliorate the effects. We investigated this issue in a double transgenic rat model (dTGR) that produces copious amounts of Ang II locally [3,4]. We gave the animals cerivastatin, 0.5 mg/kg, daily for 3 weeks by gavage. Both NF-{kappa}B and AP-1 transcription factor activation in the kidney, as shown by electrophoretic mobility shift assays, were significantly reduced with statin treatment (Figures 1AGo and 2Go). Immunostaining for the p65 NF-{kappa}B component was similarly reduced (Figure 1BGo) as was c-fos mRNA expression (Figure 2Go).



View larger version (105K):
[in this window]
[in a new window]
 
Fig. 1.  (A) Electrophoretic mobility shift assay for NF-{kappa}B DNA binding from whole kidney. Cerivastatin treatment reduced binding. (B) Co-localization of bFGF, IL-6, and the NF-{kappa}B component p65. Pink staining for all three is seen in dTGR vessel, but not in cerivastatin-treated or SD vessels.

 


View larger version (101K):
[in this window]
[in a new window]
 
Fig. 2.  Electrophoretic mobility shift assay for AP-1 DNA binding from whole kidney. c-fos and c-jun mRNA expression was determined separately by RT–PCR and quantitated. c-fos expression was reduced.

 
In the heart, the effects were no less dramatic. Cerivastatin treatment reduced cardiac hypertrophy. Cerivastatin treatment also reduced extracellular matrix deposition. Fibronectin and laminin staining were decidedly less in the hearts of statin-treated dTGR compared with SD rats. NF-{kappa}B and AP-1 activation were reduced to a similar degree as in the kidneys. Untreated dTGR show markedly increased expression of the interleukin (IL)-6 and basic fibroblast growth factor (bFGF) in the media of coronary vessels, which was reduced by cerivastatin treatment. bFGF and IL-6 were also present in the perivascular space and between the myofibrils. With cerivastatin treatment, the staining for both substances was reduced, as was macrophage infiltration (Figure 3Go).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.  Immunohistochemical staining for bFGF, IL-6, and macrophage infiltration. Inserts show confirmatory studies and quantification.

 

Statins block the response to Ang II

We did not do a detailed analysis of signal transduction in these studies. However, we were able to show that activation of the extracellularly regulated kinase (ERK) was highly activated in dTGR and that this activation was reduced by statin treatment. We also tested this response in cultured vascular smooth muscle cells and showed that exposing the cells to Ang II resulted in ERK phosphorylation. This response was abolished by 30 min pre-treatment of the cells with cerivastatin. When mevalonate was given to circumvent the statin effect, the ERK phosphorylation was no longer inhibited, indicating that the action does indeed involve 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoA) reductase inhibition.

The rationale for employing statins to ameliorate Ang II-induced vascular injury comes from various sources [5]. In cell culture experiments that are clearly independent of any low-density lipoprotein cholesterol-dependent (LDL) effects, HMG-CoA reductase inhibition was effective in blocking platelet-derived growth factor and Ang II-mediated induction of c-jun and c-fos, components of AP-1 [6]. Vascular smooth muscle cells were also exposed to phorbol ester in the presence of the HMG-CoA reductase inhibitor lovastatin. Phorbol ester-induction of AP-1 activation was inhibited, suggesting that protein kinase C (PKC) signalling is also influenced by HMG-CoA reductase inhibition. The protection was blocked by the concomitant addition of mevalonate, farnesylpyrophosphate, and geranylgeranyl pyrophosphate suggesting that the mechanisms indeed involved inhibition of mevalonate synthesis by lovastatin. In a rat study of aortic banding, simvastatin was successful in reducing left ventricular hypertrophy almost to the same degree as an ACE inhibitor [7]. Furthermore, hydroxyproline deposition, tissue ACE activity, and vascular Ang II content were reduced. Clinical data also suggest that statins may modulate the renin–angiotensin–aldosterone system. Nickenig et al. [8] recently showed that hypercholesterolaemic men have greater hypertensive responses to infused Ang II and high AT-1 receptor expression compared with normocholesterolaemic men. Statin treatment rapidly reversed the exaggerated response to Ang II infusion and led to a down-regulation of AT-1 receptors.

Our findings, that Ang II-induced NF-{kappa}B activation can be reduced by statins fits well with those of Ortego et al. [9] who found that atorvastatin inhibited NF-{kappa}B activation induced by TNF-{alpha}. As a result, surface adhesion molecule expression, inflammatory infiltration, tissue factor production, matrix protein production, and cellular proliferation were all attenuated. The same group had reported similar findings with red wine earlier [10]. Possibly additive effects could be achieved. However, this hypothesis has not yet been tested.

The role of prenylation of Ras superfamily proteins

The mechanisms may involve G proteins involved in receptor-coupled signal transduction, particularly Rho. The role of RhoA/Rho-kinase in vascular biology has recently been reviewed [11]. The Rho proteins belong to the Ras superfamily. The Ras proteins alternate between an inactivated GDP-bound form and activated GTP-bound form, allowing them to act as molecular switches for growth and differentiation signals. Prenylation is a process involving the binding of hydrophobic isoprenoid groups consisting of farnesyl or geranylgeranyl residues to the C-terminal region of Ras protein superfamily. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate are metabolic products of mevalonate that are able to supply prenyl groups. The prenylation is conducted by prenyl transferases. The hydrophobic prenyl groups are necessary to anchor the Ras superfamily proteins to intracellular membranes so that they can be translocated to the plasma membrane [12]. The final cell-membrane fixation is necessary for Ras proteins to participate in their specific interactions. Statins decrease the production of mevalonate, geranyl pyrophosphate, and farnesyl pyrophosphate, and subsequent products on the way to construction of the cholesterol molecule. Thus, statins could act, independently of circulating LDL, by intracellularly interfering with Ras superfamily protein function. An abbreviated schema is provided (Figure 4Go) [13]. Ikeda et al. [14] recently showed that statins decrease matrix metalloproteinase-1 expression through inhibition of Rho. Laufs et al. [15] showed that suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4.  Prenylation of Ras GTPases. Conceivably, statin treatment attenuates the production of prenyl groups necessary for G-protein anchoring into cell membranes.

 

Effects of statins on endothelial cell function

Other novel statin-related effects, independent of extracellular cholesterol, have been described recently. Feron et al. [16] observed that atorvastatin reduced caveolin-1 abundance in endothelial cells, irrespective of whether or not LDL cholesterol was present in the medium. Atorvastatin also restored the agonist-induced eNOS activity in the cells. These findings may explain how endothelial dysfunction is restored by statins. The atorvastatin-related effects were completely restored by the addition of mevalonate. The caveolin-1 gene contains sterol response elements in its promoter, consistent with these findings. Scalia et al. [17] reported that simvastatin exerts anti-inflammatory effects in apolipoprotein (Apo) E-deficient mice, without lowering their serum cholesterol concentrations. Similar to our findings, leukocyte–endothelial cell relationships were restored. Furthermore, NO production was increased in the Apo E-deficient mice.

Potential human relevance?

Whether or not the animal findings we report, or the in vivo and in vitro findings observed by others, have clinical importance is unknown. Clinically, the vasculoprotective effects of lipophilic and lipophobic statins appear similar. The salubrious protective effects of a statin that is confined to acting in hepatocytes, has been well documented in clinical studies [18]. Clinical studies are now in progress to resolve these issues.

Notes

Correspondence and offprint requests to: Friedrich C. Luft, Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine, Wiltberg Strasse 50, D-13125 Berlin, Germany. Email: luft{at}fvk\|[hyphen]\|berlin.de Back

References

  1. Davignon J, Laaksonen R. Low-density lipoprotein-independent effects of statins. Curr Opin Lipidol1999; 10: 543–559[ISI][Medline]
  2. Tsunekawa T, Hayashi T, Kano H et al. Cerivastatin, a hydroxymethylglutaryl coenzyme a reductase inhibitor, improves endothelial function in elderly diabetic patients within 3 days. Circulation2001; 104: 376–379[Abstract/Free Full Text]
  3. Park JK, Muller DN, Mervaala EM et al. Cerivastatin prevents angiotensin II-induced renal injury independent of blood pressure- and cholesterol-lowering effects. Kidney Int2000; 58: 1420–1430[ISI][Medline]
  4. Dechend R, Fiebeler A, Park JK et al. Amelioration of angiotensin II-induced cardiac injury by a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor. Circulation2001; 104: 576–581[Abstract/Free Full Text]
  5. Faggiotto A, Paoletti R. State-of-the-art lecture. Statins and blockers of the renin–angiotensin system: vascular protection beyond their primary mode of action. Hypertension1999; 34: 987–996[Abstract/Free Full Text]
  6. Kreuzer J, Watson L, Herdegen T et al. Effects of HMG-CoA reductase inhibition on PDGF- and angiotensin II-mediated signal transduction: suppression of c-Jun and c-Fos in vascular smooth muscle cells in vitro. Eur J Med Res1999; 4: 135–143[Medline]
  7. Luo JD, Zhang WW, Zhang GP et al. Simvastatin inhibits cardiac hypertrophy and angiotensin-converting enzyme activity in rats with aortic stenosis. Clin Exp Pharmacol Physiol1999; 11: 903–908
  8. Nickenig G, Baumer AT, Temur Y et al. Statin-sensitive dysregulated AT1 receptor function and density in hypercholesterolemic men. Circulation1999; 100: 2131–2134[Abstract/Free Full Text]
  9. Ortego M, Bustos C, Hernandez-Presa MAJ et al. Atorvastatin reduces NF-{kappa}B activation and chemokine expression in vascular smooth muscle cells. Atherosclerosis1999; 147: 253–261[ISI][Medline]
  10. Blanco-Colio LM, Valderrama M, Alvarez-Sala LA et al. Red wine intake prevents nuclear factor-{kappa}B activation in peripheral blood mononuclear cells of healthy volunteers during postprandial lipemia. Circulation2000; 102: 1020–1026[Abstract/Free Full Text]
  11. Chitaley K, Weber DS, Webb RC. RhoA/Rho-kinase, vascular changes and hypertension. Curr Rep Hypertens2001; 3: 139–144
  12. Magee T, Marshall C. New insights into the interaction of Ras with the plasma membrane. Cell1999; 98: 9–12[ISI][Medline]
  13. Khwaja A, O'Connolly J, Hendry BM. Prenylation inhibitors in renal disease. Lancet2000; 355: 741–744[ISI][Medline]
  14. Ikeda U, Shimpo M, Ohki RK et al. Fluvastatin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells. Hypertension2000; 36: 325–329[Abstract/Free Full Text]
  15. Laufs U, Endres M, Custodis F et al. Suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho GTPase gene transcription. Circulation2000; 102: 3104–3110[Abstract/Free Full Text]
  16. Feron O, Dessy C, Desager JP, Balligand JL. Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation2001; 103: 113–118[Abstract/Free Full Text]
  17. Scalia R, Gooszen ME, Jones SP et al. Simvastatin exerts both anti-inflammatory and cardioprotective effects in apolipoprotein e-deficient mice. Circulation2001; 103: 2598–2603[Abstract/Free Full Text]
  18. Crisby M, Nordin-Fredriksson G, Shah PK, Yano J, Zhu J, Nilsson J. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation2001; 103: 926–933[Abstract/Free Full Text]