Statins enhance clonal growth of late outgrowth endothelial progenitors and increase myocardial capillary density in the chronically ischemic heart

Abstract

Background: Coronary artery disease and ischemic heart disease are leading causes of heart failure and death. Reduced blood flow to heart tissue leads to decreased heart function and symptoms of heart failure. Therapies to improve heart function in chronic coronary artery disease are important to identify. HMG-CoA reductase inhibitors (statins) are an important therapy for prevention of coronary artery disease, but also have non-cholesterol lowering effects. Our prior work showed that pravastatin improves contractile function in the chronically ischemic heart in pigs. Endothelial progenitor cells are a potential source of new blood vessels in ischemic tissues. While statins are known to increase the number of early outgrowth endothelial progenitor cells, their effects on late outgrowth endothelial progenitor cells (LOEPCs) and capillary density in ischemic heart tissue are not known. We hypothesized that statins exert positive effects on the mobilization and growth of late outgrowth EPCs, and capillary density in ischemic heart tissue.

Methodology/principal findings: We determined the effects of statins on the mobilization and growth of late outgrowth endothelial progenitor cells from pigs. We also determined the density of capillaries in myocardial tissue in pigs with chronic myocardial ischemia with or without treatment with pravastatin. Pravastatin therapy resulted in greater than two-fold increase in CD31+ LOEPCs versus untreated animals. Addition of pravastatin or simvastatin to blood mononuclear cells increased the number of LOEPCs greater than three fold in culture. Finally, in animals with chronic myocardial ischemia, pravastatin increased capillary density 46%.

Conclusions: Statins promote the derivation, mobilization, and clonal growth of LOEPCs. Pravastatin therapy in vivo increases myocardial capillary density in chronically ischemic myocardium, providing an in vivo correlate for the effects of statins on LOEPC growth in vitro. Our findings provide evidence that statin therapy can increase the density of capillaries in the chronically ischemic heart.

Figure 1. Characterization of porcine late outgrowth endothelial progenitor cells (LOEPCs).

Panel A: Phase contrast image of porcine LOEPC colony (upper image 2X magnification, lower image 10 x magnification, scale bar 500 microns). Panel B: Representative microscopic fields of LOEPCs are shown illustrating antigens characteristic of LOEPCs and endothelial cells; PECAM (CD31), VE-Cadherin (CD144), VEGFR2 (FLK1/KDR), c-Kit (CD117), endothelial specific nitric oxide synthase (eNOS), and Tie 2. Scale bars = 500 um. Results shown are representative of at least three independent experiments from separate animals.

Figure 2. Flow cytometry analysis of porcine late outgrowth endothelial progenitor cells (LOEPCs).

Representative flow cytometry plots of cultured late outgrowth endothelial progenitor cells expanded from initial clonal outgrowths from blood mononuclear cells. LOEPCs were analyzed with mouse monoclonal antibodies to the following antigens: Mouse IgG1, Mouse anti-pig CD31, CD105, CD133, and CD45. Primary antibodies were detected with Alexa Fluor 488 conjugated donkey anti-mouse antibodies. Results are representative of at least three independent experiments from separate animals.

Figure 3. Oral Pravastatin Promotes the Outgrowth of LOEPCs from Blood Mononuclear Cells.

Pigs were treated with a placebo or pravastatin (4 mg/kg orally) daily for three consecutive days. Blood mononuclear cells were obtained on day three, and cultured on type I collagen coated dishes to derive LOEPCs. The quantity of LOEPCs per 3×10⁷MNCs plated was determined using flow cytometry to determine the number of PECAM positive cells after 14 days in culture. The data are presented as the means ± SEM from eight different animals. * p<0.01 vs. control using a paired t-test.

Figure 4. In Vitro Pravastatin and Simvastatin Promotes Outgrowth of LOEPCs from Blood Mononuclear Cells.

Mononuclear cells were obtained from normal young pigs and cultured on type I collagen to derived LOEPCs. The quantity of LOEPCs per 3×10⁷MNCs plated was determined using flow cytometry for PECAM positive cells after 14 days in culture. The data are presented as the means ± SEM from eight different animals. * p<0.05, ** p<0.01 vs. control using a paired t-test.

Figure 5. In Vitro Pravastatin and Simvastatin Promotes Growth of LOEPCs under Clonogenic Conditions.

LOEPC cell lines from pigs were plated under limiting dilution conditions in the absence or presence of pravastatin or simvastatin. The number of LOEPCs was determined after seven days of growth with or without statin. The data are presented as the means ± SEM from eight independent experiments.

Figure 6. Clonogenic Growth of LOEPCs is Sensitive to AKT inhibition, while the Effect of Pravastatin and Simvastatin on LOEPC Clonogenic Growth is Not.

Panel A-Western blot analysis showing both pravastatin and simvastatin activate AKT phosphorylation, and AKT inhibitor IV blocks the phosphorylation of AKT in LOEPCs. Panel B-LOEPCs were grown under clonogenic conditions with or without an AKT inhibitor (100 nM), in the presence or absence of pravastatin or simvastatin (100 nM each) to determine if the effect of statins on LOEPC clonogenic growth as sensitive to AKT inhibition. n = 6 independent experiments. * p<0.05 vs. No Akt inhibitor IV.

Figure 7. Statins Differentially Affect Angiogenesis of LOEPCs in Vitro.

Panel A: Representative photomicrographs of porcine LOEPCs plated on polymerized Matrigel for 24 hours with no drug treatment (Control), or Pravastatin, Simvastatin or Atorvastatin (100 nM each). Images are representative of three independent experiments. Scale bar = 500 microns. Panel B: Mean vessel length was measured from photomicrographs. Means +/− SEM are shown for three independent experiments. Significant differences from untreated cells (Control) are indicated by * (p<0.05). Panel C: Branch Point Determination-the number of branch points within LOEPCs plated on Matrigel was determined from each photomicrograph. The means +/− SEM are shown for three experiments. Significant differences from untreated cells (Control) are indicated by * (p<0.05).

Figure 8. Pravastatin Increases Myocardial Capillary Density in Animals with Chronic Myocardial Ischemia In Both Hibernating Myocardium and the Non-ischemic Remote Myocardium.

Panel A-Representative immunofluorescence images of myocardial tissue from untreated or Pravastatin treated pigs with hibernating myocardium. Scale bar = 500 microns. Panel B-Quantitative assessment of CD31+ cells per square millimeter in chronically ischemic hibernating myocardium in the territory of the left anterior descending artery, and the opposite non-ischemic posterolateral wall, n = 6 animals in pravastatin treated group, n = 5 non-statin treated animals, p<0.05.