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Rosuvastatin reduces experimental left ventricular infarct size after ischemia-reperfusion injury but not total coronary occlusion

¹Cardiovascular Division, Department of Medicine, Cambridge; and ²Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, and Harvard Medical School, Boston, Massachusetts

Submitted 16 September 2004 ; accepted in final form 16 November 2004

	ABSTRACT

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This study compared the effects of rosuvastatin on left ventricular infarct size in mice after permanent coronary occlusion vs. 60 min of ischemia followed by 24 h of reperfusion. Statins can inhibit neutrophil adhesion, increase nitric oxide synthase (NOS) expression, and mobilize progenitor stem cells after ischemic injury. Mice received blinded and randomized administration of rosuvastatin (20 mg·kg^–1·day^–1) or saline from 2 days before surgery until death. After 60 min of ischemia with reperfusion, infarct size was reduced by 18% (P = 0.03) in mice randomized to receive rosuvastatin (n = 18) vs. saline (n = 22) but was similar after permanent occlusion in rosuvastatin (n = 17) and saline (n = 20) groups (P = not significant). Myocardial infarct size after permanent left anterior descending coronary artery occlusion (n = 6) tended to be greater in NOS3-deficient mice than in the wild-type saline group (33 ± 4 vs. 23 ± 2%, P = 0.08). Infarct size in NOS3-deficient mice was not modified by treatment with rosuvastatin (34 ± 5%, n = 6, P = not significant vs. NOS3-deficient saline group). After 60 min of ischemia-reperfusion, neutrophil infiltration was similar in rosuvastatin and saline groups as was the percentage of CD34⁺, Sca-1⁺, and c-Kit⁺ cells. Left ventricular NOS3 mRNA and protein levels were unchanged by rosuvastatin. Rosuvastatin reduces infarct size after 60 min of ischemia-reperfusion but not after permanent coronary occlusion, suggesting a potential anti-inflammatory effect. Although we were unable to demonstrate that the myocardial protection was due to an effect on neutrophil infiltration, stem cell mobilization, or induction of NOS3, these data suggest that rosuvastatin may be particularly beneficial in myocardial protection after ischemia-reperfusion injury.

nitric oxide; neutrophil; myocardial salvage; stem cells

The most compelling data with statins are those reported after ischemic injury with reperfusion, rather than with permanent coronary occlusion (9–11, 14, 30). A direct comparison of the effects of rosuvastatin between permanent coronary artery occlusion and transient ischemia followed by reperfusion has not been performed. Because of advances in early diagnosis and interventional techniques, a growing proportion of acute MI patients are treated with early reperfusion. We therefore performed a randomized and blinded evaluation of the effect of rosuvastatin on left ventricular (LV) infarct size and remodeling after permanent ischemia vs. transient, 60-min ischemia with reperfusion to establish the effect of rosuvastatin on infarct size in these two experimental model systems. We tested the hypothesis that rosuvastatin decreases infarct size in the reperfused myocardium in association with a reduction of neutrophil accumulation in the injured myocardium, an increase in NOS3 expression, and mobilization of stem cells.

	MATERIALS AND METHODS

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Randomization of mice, rosuvastatin treatment, and method of blinding. Animal studies were conducted in conformity with a protocol approved by the Harvard Standing Committee on Animals. Male C57BL/6 mice, 10–14 wk of age, were randomized to receive once-daily subcutaneous injections of 20 mg·kg^–1·day^–1 rosuvastatin dissolved in saline or saline alone. This dose was selected because it has previously been shown to reduce cerebral infarct size after ischemia-reperfusion (13). Dosing was begun 2 days before surgery and continued until the time of death. Rosuvastatin or saline was coded so that investigators performing all analyses were blinded, and coding was revealed on completion of sample processing and analyses.

Coronary artery ligation. Mice were anesthetized with pentobarbital sodium (90 mg/kg ip). A tracheotomy was performed via a midline cervical incision with a PE-90 endotracheal tube, and mechanical ventilation with room air was begun at 0.3–0.4 ml at 100 rpm (Harvard Apparatus). A thoracotomy was performed to expose the heart. For permanent coronary artery occlusion, the left anterior descending artery (LAD) was visualized and ligated with 7-0 silk suture as previously described (22). For ischemia-reperfusion, the LAD was visualized and ligated with 7-0 silk suture with a slip knot to allow for reperfusion. The LV was reperfused after 60 min, and the ligature was kept in place for later determination of LV area at risk. Animals were placed on a heating pad for 3 h during recovery, and buprenorphine (0.03 mg im) was administered twice a day for pain relief until the time of death. A 13-MHz linear probe (Sequoia, Siemens, Mountain View, CA) was used for echocardiograms, which were performed 2 and 28 days after permanent coronary artery ligation. Mice were then euthanized, and the LV was harvested, weighed, and fixed in 4% formaldehyde. After paraffin fixation, consecutive slices were obtained, and MI size was measured (22). Tail cuff blood pressure was measured as described elsewhere (23).

Determination of LV infarct size after ischemia-reperfusion. Mice were anesthetized with intraperitoneal pentobarbital, and the heart was excised and mounted on an isolated heart perfusion apparatus. The isolated heart was perfused at 37°C and 60 mmHg retrograde through the aorta with Krebs buffer followed by 1% 2,3,5-triphenyltetrazolium chloride solution for 10 min to stain viable myocardium. The coronary artery was reoccluded with the suture that was left in place at the time of reperfusion. The heart was then perfused with filtered 10% phthalocyanine blue in phosphate-buffered saline to define the LV area at risk. The LV was sliced in cross section into six sections and fixed in 4% buffered paraformaldehyde. Each section was weighed and photographed. Nonjeopardized (region outside the ligated area) LV tissue was identified by deep blue staining, ischemic but viable myocardium was identified by deep red staining, and necrotic LV tissue was identified by white coloration. A planimetry image analysis program developed in our laboratory was used by an investigator blinded to treatment group to quantitate the three regions in each slice. The areas of the three regions from all slices were summed to calculate their respective volumes. Infarct size is expressed as percentage of LV volume at risk.

Neutrophil and leukocyte immunohistochemistry. Rosuvastatin- and saline-treated mice underwent ischemia-reperfusion surgery as described above and were killed 1 day [rosuvastatin (n = 8) and saline (n = 5)] and 2 days [rosuvastatin (n = 5) and saline (n = 8)] after reperfusion. Mice were anesthetized with intraperitoneal pentobarbital, and the heart was excised, rinsed in phosphate-buffered saline, fixed in buffered 4% paraformaldehyde, and sliced into six sections as described above. The fifth slice from the base of the LV was embedded in paraffin. Immunohistochemistry on 7-µm sections was performed with rat monoclonal anti-mouse neutrophil MCA771G antibody (Serotec, Oxford, UK) and leukocyte common antigen (CD45, eBioscience, San Diego, CA) as primary antibodies and anti-rat IgG biotinylated monoclonal antibody as secondary antibody. Staining was generated with the Vectastain ABC Kit Peroxidase Rat IgG PK-4004 (Vector Laboratories). Positively stained cells from the infarcted myocardium were counted by an investigator blinded to treatment group.

Peripheral blood flow cytometry. At 24 h after reperfusion at the time of ex vivo heart perfusion, blood was collected from the inferior vena cava of rosuvastatin-treated (n = 5) and saline-treated (n = 5) mice. Whole blood samples were blocked with mouse CD16/32 for 10 min and stained for 30 min on ice with anti-mouse phycoerythrin (PE)-c-Kit (eBiosciences), FITC-CD34 (eBiosciences), or FITC-Sca-1 as stem cell markers and PE- or FITC-conjugated IgG antibodies as isotope controls (eBiosciences). Red blood cells were lysed, and samples were washed twice in flow cytometry buffer. Gating on the PE or FITC channel was set at 1–3% on the isotype control samples, and the percentages of positive-staining cells were determined for each sample. Samples were run in duplicate.

LV NOS3 mRNA and protein determination. TriReagent (Sigma) was used to extract RNA from LV and lung tissue and Bio-Rad Protein Extraction Reagent (Bio-Rad) was used to extract protein from LV tissue of mice treated with rosuvastatin (n = 3–5) or saline (n = 3–5) for 5 days. NOS3 mRNA levels were determined by Northern blotting with a ³²P-labeled cDNA fragment of mouse NOS3 (GenBank accession no. BC052636). Quantitative real-time PCR was used for quantitation of NOS3 RNA levels using CybrGreen (Qiagen) with the LightCycler system (Roche). Primers amplified a 176-nt product (nt 1157–1317) from mouse NOS3 (GenBank accession no. BC052636). Data were normalized to mouse tubulin mRNA levels. NOS3 protein levels were determined using Western analysis with anti-mouse NOS3 antibody (Lab Vision).

Statistical analysis. Values are means ± SE. Data were analyzed by Student's t-test, except in Fig. 4B, where data were analyzed by analysis of variance.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Nitric oxide synthase isoform 3 (NOS3) mRNA and protein levels in rosuvastatin- and saline-treated mice. A: NOS3 protein determined by Western blotting (top) and NOS3 mRNA determined by Northern blotting (bottom) show that rosuvastatin had no effect on LV NOS3 protein and mRNA levels compared with samples from saline-treated mice. EtBr, ethidium bromide. B: quantification of NOS3 [endothelial NOS (eNOS)] mRNA levels in LV samples from rosuvastatin- and saline-treated mice. Quantitative RT-PCR confirmed that rosuvastatin had no effect on NOS3 mRNA levels. Data were normalized to -tubulin levels.

	RESULTS

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View larger version (48K): [in this window] [in a new window]   Fig. 1. A: left ventricular (LV) internal dimensions, fractional shortening, and infarct size in rosuvastatin- and saline-treated mice with permanent coronary artery ligation. Rosuvastatin had no effect on LV internal dimensions, fractional shortening, or infarct size compared with saline-treated mice. B: LV area at risk (AAR) and infarct size in rosuvastatin- and saline-treated mice with transient (60 min) myocardial ischemia followed by reperfusion. Rosuvastatin decreased infarct size by 18% compared with saline-treated mice. Representative stained hearts used for measurements of infarct size are also shown. NS, not significant.

Rosuvastatin and infarct size after ischemia and reperfusion. We hypothesized that rosuvastatin may be preferentially protective after 60 min of ischemia and reperfusion compared with total occlusion given the participation of inflammation in ischemia-reperfusion. Therefore, we performed a blinded and randomized evaluation of rosuvastatin on infarct size. At 1 day after reperfusion, MI size was significantly reduced by 18% in mice treated with rosuvastatin compared with those treated with saline: 46 ± 4% vs. 56 ± 3% (P = 0.03; Fig. 1B). Area at risk was similar in saline-treated (n = 22) and rosuvastatin-treated (n = 18) mice (38 ± 3 and 43 ± 3, respectively, P = NS), indicating that placement of the ligature was similar between the groups (Fig. 1B). This demonstrates that rosuvastatin decreases the extent of myocardial necrosis when the myocardium is reperfused after coronary artery occlusion.

Rosuvastatin and leukocyte infiltration after ischemia and reperfusion. The beneficial effect of rosuvastatin, i.e., reduction of infarct size after ischemia-reperfusion, may be due to its regulation of early leukocyte and neutrophil infiltration to the injured myocardium (9). The number of neutrophils in the injured myocardium increased after ischemia-reperfusion but was similar between rosuvastatin- and saline-treated mice 1 day (465 ± 131 vs. 527 ± 136 neutrophils/section, P = NS) and 2 days (403 ± 71 vs. 438 ± 57 neutrophils/section, P = NS) after reperfusion (Fig. 2A). Similarly, the number of cells staining positive for leukocyte common antigen (CD45) in the injured myocardium increased after ischemia-reperfusion and was similar in rosuvastatin- and saline-treated mice 1 day (112 ± 30 vs. 99 ± 23 leukocytes/section, P = NS) and 2 days (306 ± 67 vs. 341 ± 34 leukocytes/section, P = NS) after reperfusion (Fig. 2B). These data suggest that the beneficial effect of rosuvastatin on infarct size after ischemia-reperfusion is not related to an inhibitory effect of rosuvastatin on leukocyte and neutrophil aggregation and adhesion in injured myocardium.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Rosuvastatin had no effect on neutrophil (A) and leukocyte (B) infiltration into the myocardium after ischemia-reperfusion.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Progenitor/stem cell staining in peripheral blood from rosuvastatin- and saline-treated mice. Staining for CD34, Sca-1, and c-Kit was similar in rosuvastatin- and saline-treated mice.

	DISCUSSION

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Progressive LV remodeling after MI leads to deterioration in contractile function associated with increased morbidity and mortality. Reperfusion strategies to restore blood flow limit ventricular remodeling after MI by salvaging jeopardized myocardium, which decreases the incidence of congestive heart failure and improves survival. In this study, we compared the effects of rosuvastatin on infarct size in the permanent occlusion and ischemia-reperfusion models of MI. An important finding of the present study is that rosuvastatin had no beneficial effect on infarct size in the permanent occlusion model but significantly decreased infarct size (necrotic area/area at risk) after 60 min of ischemia with reperfusion. The novel finding of this study is the beneficial effect of rosuvastatin in salvaging reperfused myocardium in the absence of an effect on neutrophil and leukocyte recruitment, a decrease in NOS3 expression, or stem cell mobilization, effects that have been reported in association with salvage after ischemia with statins.

Rosuvastatin is relatively hydrophilic; therefore, in contrast to lipophilic statins, it has reduced access to many cell types throughout the body by diffusion across cell membranes (18). Uptake of rosuvastatin is extremely high in hepatocytes, cells that express active transporters for anionic compounds such as rosuvastatin. This property, in association with the higher affinity of rosuvastatin for the active site of hydroxymethylglutaryl (HMG)-CoA reductase than of other statins (18), explains its selectivity for inhibition of HMG-CoA reductase in hepatocytes compared with fibroblasts (17). However, pleiotropic effects of rosuvastatin have clearly been documented. For example, Susic et al. (26) demonstrated that rosuvastatin reduced hypertension in genetically hypertensive rats independent of effects on cholesterol. Furthermore, rosuvastatin can correct nerve and vascular function in diabetic mice, also independent of effects on cholesterol (20). Intriguingly, in this study, cotreatment with mevalonate inhibited the beneficial effects of rosuvastatin, suggesting that, at least in this study, the benefit was independent of cholesterol lowering but via effects dependent on cholesterol biosynthesis pathway inhibition (20). This may explain why rosuvastatin has high selectivity for hepatocyte HMG-CoA reductase but still clearly has powerful pleiotropic effects relevant to cardioprotection (Fig. 5), such as the infarct size reduction seen in this study.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Schematic of potential mechanisms of statins in cardioprotection during ischemia and reperfusion. ROS, reactive oxygen species.

Although the degree of neutrophil accumulation in the ischemic-reperfused myocardium was not altered by rosuvastatin treatment, rosuvastatin may have altered neutrophil function, for example, through an attenuation of neutrophil generation of reactive oxygen species (ROS) (4). Statins decrease NADPH oxidase-related ROS generation through inhibition of prenylation and translocation of cytosolic rac1 to the membrane subunits of NADPH oxidase. Lipophilic and hydrophilic statins have this effect in the heart (16). Thus, because ROS generation during ischemia-reperfusion contributes to cell death, the beneficial effect of rosuvastatin on myocyte death and infarct size may have been through a decrease in neutrophil and/or myocyte ROS generation early after activation following ischemia-reperfusion (2). Results from animal studies have shown that neutrophil depletion has beneficial effects in preventing ischemia-reperfusion injury. However, neutrophil depletion in humans has not been very successful (21; for review see Ref. 28), probably because neutrophils also have beneficial functions in promoting healing and scar formation, and the balance between beneficial and detrimental effects may differ in animal models and humans. Statins may favorably alter neutrophil function to allow their beneficial effects to dominate in the setting of ischemia and reperfusion.

Statins can induce the differentiation of endothelial progenitor cells from peripheral blood mononuclear cells cultured under endothelial cell growth conditions through a mechanism involving phosphatidylinositol 3-kinase/Akt signaling similar to vascular endothelial growth factor (VEGF) (3, 15). Atorvastatin has been shown to increase the expression of VEGF receptor 2 (KDR) on peripheral blood CD34 cells without increasing the total number of CD34⁺ cells, demonstrating the potential for atorvastatin to induce the differentiation of peripheral blood progenitor cells toward an endothelial cell phenotype (27). The number of bone marrow-derived cells that also stained for endothelial markers was increased by simvastatin, which was associated with accelerated reendothelialization and significant reductions in neointimal thickening in balloon-injured arterial segments in rats (29). Cultured splenocytes and bone marrow cells harvested from mice fed simvastatin for 3 wk showed an increase in the number of cells staining positive for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated LDL/lectin compared with saline-treated mice (29). In the present study, we did not observe an increase in the number of peripheral blood cells staining positive for CD34, c-Kit, and Sca-1 in response to rosuvastatin with the dosing regimen used. However, we cannot rule out the possibility that expansion in culture under endothelial cell growth conditions is required to observe this effect with rosuvastatin. Furthermore, because studies have shown in humans and mice that longer dosing times with statins are required to achieve bone marrow mobilization of cells that differentiate into the endothelial cell phenotype, this effect is unlikely to account for the reduction in infarct size by rosuvastatin in the present study. Recent studies have shown that peripheral blood cells expressing the monocyte marker CD14 (CD14⁺ cells that are usually CD34^–) may function as pluripotent progenitor cells able to differentiate into endothelial cells when cultured with VEGF, as well as into macrophages, epithelial cells, neuronal cells, and liver cells when cultured with appropriate growth factors (7, 8, 32). The differentiation of CD14⁺ cells into specific lineage cells is accompanied by a decrease in CD14 expression and an increase in lineage markers. Simvastatin treatment in patients with hypercholesterolemia was associated with a decrease in CD14 expression in peripheral blood (25). It is attractive to consider the possibility that statins can induce this special subset of CD14⁺ peripheral blood cells to differentiate into endothelial progenitors as a mechanism contributing to improved endothelial function with statins. Peripheral blood CD14 expression was not measured in the present study, and a possible beneficial effect of statins on this phenomenon needs to be resolved in future studies.

One of the most characterized effects of statins independent of cholesterol lowering is the ability to increase the expression and activity of eNOS (NOS3). Absence of NOS3 in mice subjected to permanent coronary artery ligation is associated with increased mortality, greater myocardial remodeling, decreased fractional shortening, and impaired diastolic function compared with wild-type mice, effects that are not prevented by normalization of blood pressure in NOS3-deficient mice (23). In the ischemia-reperfusion model, NOS3 deficiency was associated with greater infarct size and more neutrophil infiltration in the postischemic myocardium (10). These effects were attributed to decreased bioavailability of NO in NOS3-deficient mice, resulting in an increased expression of endothelial adhesion molecules and a greater inflammatory response leading to necrosis. Statins inhibit leukocyte adhesion and promote eNOS production by inhibiting Rho kinase geranylgeranylation and, possibly, by directly activating eNOS expression and activity through activation of Akt (24). In contrast to several reports describing an increase in eNOS mRNA expression and activity with statin treatment in the ischemia-reperfusion model in association with a reduction in infarct size, we found no increase in eNOS mRNA and protein, although infarct size was significantly decreased in mice treated with rosuvastatin. Although we cannot explain this discrepancy, it is possible that local increases in NO production in the endothelium due to statin treatment that are not detected in whole tissue extracts contributed to the reduction in infarct size after ischemia-reperfusion in our model. In support of this, infarct size in NOS3-deficient mice in our study tended to be greater than in wild-type mice, in agreement with a published report (10).

It is well accepted that, with regard to damage after ischemia and reperfusion, timing is critical. The earlier the myocardium is reperfused after ischemia, the greater the myocardial salvage; conversely, the longer the period of ischemia, the less the myocardial salvage. In general, longer periods of ischemia lead to greater necrosis, rather than apoptosis, a form of cell death that is amenable to therapeutic intervention and prevention. An increase in neutrophil accumulation after 30 min vs. 20 min was observed after the same duration of reperfusion by Jones et al. (10). Apoptosis after ischemia-reperfusion measured by in vivo annexin staining in mouse hearts was also increased after 30 min vs. 15 min of ischemia (5). This same study also showed that apoptosis during reperfusion increases to an extent dependent on the time of ischemia. In the present study involving ischemia and reperfusion, we used 60 min of ischemia followed by 24 h of reperfusion. This duration of ischemia was within the critical time window for myocardial salvage by rosuvastatin, whereas no beneficial effect of rosuvastatin was observed in the group subjected to permanent ligation. Additional studies are required to determine the duration of time of both ischemia and reperfusion for which rosuvastatin has a beneficial effect on infarct size as well as on the additional end points that were measured in the present study.

In summary, rosuvastatin had no beneficial effect on infarct size after permanent coronary artery ligation. In contrast, rosuvastatin treatment resulted in a significant decrease in infarct size after 60 min of ischemia-reperfusion. The decrease in infarct size was observed in the absence of an effect of rosuvastatin on neutrophil/leukocyte infiltration, progenitor cell mobilization, and eNOS mRNA and protein expression. Despite its limited bioavailability, rosuvastatin treatment of reperfused myocardium after ischemic injury decreased myocardial cell death. The protective effect of rosuvastatin may have been on apoptotic cell death, because the predominant form of cell death early after ischemia-reperfusion injury is apoptotic, and this form of cell death is amenable to reduction.

	GRANTS

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This study was funded in part by research grants from the National Institutes of Health and in part by a research grant from AstraZeneca.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. T. Lee, Cardiovascular Div., Brigham and Women's Hospital, 65 Landsdowne St., Cambridge, MA 02139 (E-mail: rlee{at}rics.bwh.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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