Statins have become a cornerstone of risk modification for ischaemic heart disease patients. A number of studies have shown that they are effective and safe. However studies have observed an early benefit in terms of a reduction in recurrent infarct and or death after a myocardial infarction, prior to any significant change in lipid profile. Therefore, pleiotropic mechanisms, other than lowering lipid profile alone, must account for this effect. One such proposed pleiotropic mechanism is the ability of statins to augment both number and function of endothelial progenitor cells. The ability to augment repair and maintenance of a functioning endothelium may have profound beneficial effect on vascular repair and potentially a positive impact on clinical outcomes in patients with cardiovascular disease. The following literature review will discuss issues surrounding endothelial progenitor cell (EPC) identification, role in vascular repair, factors affecting EPC numbers, the role of statins in current medical practice and their effects on EPC number.

The maintenance of endothelial integrity is essential for the preservation of a healthy vasculature[1]. This integrity results from a balance between on-going endothelial damage and the rate of vascular repair. Disruption of endothelial integrity or impairment of endothelial repair mechanisms is a central step in both the initiation and progression of atherosclerosis[2]. Endothelial repair is dependent on undifferentiated cells migrating to sites of vascular injury[3-5] then differentiating into mature endothelial cells[6-13]. These undifferentiated cells are called endothelial progenitor cells (EPCs) have a central role in vascular repair by virtue of their ability to proliferate, migrate to site of vascular injury and then differentiate into mature vascular endothelium[13,14]. EPCs perpetuate this cycle by secreting pro-angiogenic cytokines[15].

Statins form the corner stone of treatment of coronary artery disease. The safety and efficacy of statins in reducing cardiac events by decreasing serum LDL-cholesterol has been well described[16-18]. Recently however studies have shown the early beneficial effect of statins occurs before any significant change in lipid profile. This led to the hypothesis that cardiovascular benefits of statins may occur via alternative mechanisms other than reduction of LDL-cholesterol alone[19,20]. One such proposed mechanism is the ability of statins to augment both number and function of EPCs.

The following literature review discusses issues surrounding EPC identification, role in vascular repair, factors affecting EPC numbers, the role of statins in current medical practice and their effects on EPC number.

We performed a review of various studies within the literature available on endothelial progenitor cell and statins. The authors searched various databases (EMBASE, OVID, PubMed) using the keywords: “Endothelial progenitor cells”, “statin”, “pleiotropic effects”. We studied the various publications that we obtained from the search results. Full text manuscripts were obtained. We only included papers in the English language.

Cellular identification and staging of differentiation has been made possible by specific surface receptors called epitopes that allow immunophenotyping. This process allows identification of subset of cellular surface molecule termed cluster of differentiation (CD). Cellular subtypes may be defined by the presence or absence of a particular CD molecule. Therefore “CD” may be “+ “or “-” denoting either presence or absence of a particular CD, and is used to describe stem cells rather than fully differentiated cell types. Certain cell types may have variable CD marker expression during maturation for example, and therefore classed as bright (high), mid (mid) or dim (low) denoting intensity of expression[21,22].

Vascular repair had previously been thought to be due to migration and proliferation of fully differentiated endothelial cells, in a process called angiogenesis[23]. Asahara et al[24] identified putative cells with cell surface marker CD34⁺, alternately named kinase insert domain receptor (KDR/VEGFR) markers capable of differentiating into endothelial cells both in vitro and in vivo[24-26]. Subsequent studies recognised that in fact undifferentiated cells subsequently termed EPCs migrated to sites of neovascularization and then differentiate into endothelial cells[24,26] in a process called vasculogenesis[27]. EPCs are derived from pluripotential stem cells within the bone marrow. These then evolve into mature endothelial cells[24] accounting for only 0.001%-0.0001% of peripheral blood cells in an unstressed state[28]. Circulating EPCs may be isolated from bone marrow or the circulation as mononuclear cells[24,29,30], expressing a variety of endothelial surface markers[31]. However, there currently remains a lack of consensus on phenotypic and functional definition of endothelial precursor cells[32,33].

EPCs are a diverse group of cells of different lineages that have angiogenic potential, but are not always necessarily able to differentiate into functional endothelial cells as would be suggested from their name[32]. EPCs are derived from CD34⁺ hematopoietic progenitor cells[6,24,29,31], with the subset of EPCs characterized by co-expression of endothelial marker proteins[6,29,31]. Studies have identified 3 markers associated with early functional EPCs including CD133, CD34, and the vascular endothelial growth factor receptor-2 (VEGFR-2) also known as kinase insert domain receptor (KDR, Flk-1 or CD309)[7,31]. Therefore EPCs express markers of both hematopoietic stem cells (CD34 and CD133) and endothelial cells (CD146, vWF, and VEGFR2)[26,28,29,31,34-37]. The presence of certain cell surface markers are depend on the stage of maturations of the EPC. For example the cell surface marker CD133, a 120-kDa trans-membrane polypeptide, is expressed on bone marrow derived hematopoietic stem and progenitor cells in peripheral blood[37]. The expression of CD133 on EPCs declines during maturation within the peripheral circulation. Currently there remains some uncertainty as to when EPCs lose the CD133 surface marker, whether during transmigration from bone marrow to circulation or later whilst in the peripheral blood system[38]. Nevertheless the loss of CD133 represents the transformation into more mature EPCs that are endothelial-like cells[37]. Whereas the expression of CD34 a cell surface marker found on immature pluripotential stem cells[31] that acts as an adhesion molecule, although the precise function remains unclear, gradually increases as the CD133 decreases as the EPC matures[37]. During the course of maturity EPCs begin to have increased expression of other markers specific to endothelial cells such as VEGFR-2, VE-cadherin, and von Willebrand factor (vWF)[37].

The expression of CD34⁺, CD133⁺, and/or VEGF2⁺ has been used as identifying markers of EPCs in a number of studies[28,32,39-41]. Whereas as other studies advocate the use of CD133⁺ either alone or in combination with CD34⁺/VEGFR-2⁺ cells for identification of EPCs[31,42]. In contrast, other studies have suggested that CD133⁺ are haematopoietic cell lines and have not been identified in and therefore unable to form endothelial phenotypic EPCs[7,40,43]. Ingram et al[28] proposed that CD45^- cells incorporated “true” circulating EPCs and verified by other studies[28,39,40,43]. Interestingly, CD34⁺/VEGFR2⁺ and diminished (dim) CD45 (CD45^dim) cells have been found to have greater correlation with coronary heart disease severity and response to statin therapy[44,45].

In summary, the maturity of the EPC is marked by the gradual loss of CD133, gradual increased expression of CD34⁺ and the appearance of CD31, VE-cadherin and vWF cell surface markers (Table 1).

Endothelial integrity is essential for healthy vasculature, and can be thought of as a balance between continued endothelial damage and the capacity to repair by a pool of EPCs[9,46]. It is now generally accepted that cardiovascular risk correlates with EPC numbers. Highlighting the integral relationship between endothelium and atherosclerosis[47-51], disruption of endothelial integrity by endothelial cell injury has been shown to be a stimulus for the development of atherosclerosis[2], but also as a stimulus for augmentation of EPC number and function[9,52,53]. Continued endothelial damage[54] may lead to an eventual reduction of the number of EPCs. Elevated EPC numbers have been shown to be associated with augmented formation of collaterals in coronary artery disease[55] and restoration of endothelial vasodilator function[9]. A reduction in EPC numbers may lead to deficient endothelial repair and progression of atherosclerosis, with further EPC depletion and perpetuation of atherosclerosis[9,56]. However, it is uncertain whether low numbers of circulating EPCs represents enhanced usage by vascular repair processes, or reduced production by bone marrow.

CD34⁺ VEGFR2⁺ EPCs cells have been shown to be reduced in patients with atherosclerotic coronary and peripheral disease[57]. Vasa et al[56] found not only reduced numbers, but also impaired function of EPCs in patients with coronary artery disease. Elevated numbers of EPC have been associated with freedom from myocardial infarction, hospitalization, revascularization and cardiovascular death in patients with coronary artery disease[56,58]. Furthermore the predictive value of EPC count has been shown to be independent of traditional cardiovascular risk factors[9,46,59]. In fact, the extent of the reduction in EPC numbers has been associated not only with coronary artery disease burden[60], but also the presence of symptoms[61,62].

Finally, elevated numbers of circulating CD34⁺/CD133⁺/VEGFR2 EPCs have been observed after an acute myocardial infarction[42,63]. This may be regarded as a consequence of cardiac ischaemia together with raised inflammatory and haematopoietic cytokines stimulating EPC mobilisation from the bone marrow[64-66]. A similar response is seen following coronary angioplasty[67], and interestingly, the combination of an acute coronary syndrome (ACS) treated by angioplasty provoked an enhanced EPC response[68]. Therefore, EPC may have a central role not only in repairing coronary vessels after plaque rupture, but also after any coronary intervention.

Statins act by competitively inhibiting 3 hydroxy-3-methylglutaryl Coenzyme A (HMG CoA) reductase, the rate limiting step in the mevalonate pathway producing isoprenoids including cholesterol. The competitive inhibition of HMG CoA reductase induces the expression of LDL receptors within the liver, thereby increasing the catabolism of plasma LDL, with a consequent decrease in LDL-cholesterol levels[69].

The safety and efficacy of statins in reducing cardiac events by decreasing serum LDL-cholesterol has been well described[16-18]. Statin therapy has been shown to reduce death and cardiovascular events in primary prevention of atherosclerosis[70], stable coronary artery disease[16,71-73], ACS[74,75] and secondary prevention[72]. Statins also appear to reduce development of atherosclerotic lesions[76,77] and decrease plaque burden[13,78].

The beneficial effect of intensive statin therapy was studied in a prospective meta-analysis of 90056 patients from 14 randomised trials and found greater cholesterol reduction was associated with better patient outcomes[19]. The study found that the 5-year incidence of major adverse cardiac events, coronary revascularization and stroke was reduced by one fifth for every millimoles per liter reduction in LDL cholesterol, which was irrespective of the initial lipid profile[19].

Another meta-analysis found aggressive statin therapy was associated with reduced peri-procedural myocardial infarction and a 44% risk reduction in major adverse cardiovascular events at 30-d irrespective of clinical presentation[79]. Moreover, the ARMYDA-RECAPTURE study[80] found reloading of the high dose statin, atorvastatin 80 mg in 383 NSTEMI and stable angina patients on chronic therapy prior to percutaneous coronary intervention (PCI) had a 50% reduction in 30-d major adverse cardiac events in both group with a greater reduction in NSTEMI group[80].

These studies led to the universal adoption of statin therapy in patients with coronary artery disease irrespective of presentation from stable angina to ACSs[81,82].

Several studies have shown the early beneficial effect of statins occurs before any significant change in lipid profile. This led to the hypothesis that cardiovascular benefits of statins may occur via alternative mechanisms other than reduction of LDL-cholesterol alone[19,20]. These potential beneficial effect(s) may represent a potential therapeutic target for ischemic heart disease patients, and therefore is of great interest. There have been a number of mechanisms proposed to account for pleiotropic effects of statin therapy. These include reduction in vascular inflammation[83], reduction of platelet aggregability and thrombus deposition[77,84-86], enhancement of fibrinolysis[87] and increased endothelium derived NO production[88-90]. However the mechanism that has evoked the most interest is the impact of statins on EPCs[70].

Statin therapy has been associated with greater numbers of circulating EPCs by enhancing mobilization, differentiation, increasing longevity, enhance homing to sites of vascular injury with augmentation of re-endothelisation by enhancing expression of adhesion molecules on EPC cell surface membrane[3,70,91-94].

However, the duration of the effect on EPC number by statin therapy continues to remain contentious.

In one study, atorvastatin therapy was shown to significantly increase circulating EPC as soon as 1 wk with plateauing after 3-4 wk with a 3-fold increase of EPCs from baseline in a stable angina population was also observed[70]. Whereas Deschaseaux et al[95] investigated whether EPCs could be firstly detected and secondly characterized in patients receiving long-term statin therapy defined as 4 wk. The group found a significantly greater number of CD34⁺, CD34⁺/CD144⁺ circulating EPC in patients receiving statin therapy compared to statin naïve patients. Interestingly two types of EPCs were detected, early and late EPCs. The early EPCs were found to form elongated cells whereas the late EPC population gave rise to cobblestone-like colonies with strong proliferation capacities seen in-vitro cell culture. The numbers of early EPCs were significantly higher in patients not receiving statin therapy whereas late EPCs were significantly higher in patients receiving statin therapy. The study also observed that long term statin therapy specifically maintained late EPCs in circulation with a CD34⁺/CD144⁺ phenotype. Rodent studies have found rosuvastatin resulted in a greater than 3 fold increase in EPC numbers when compared with placebo as long as 10 wk after myocardial infarction[56]. Long-term atorvastatin 10 mg for 12 mo markedly increased EPC number with an associated decrease in oxidative DNA damage[35]. However to the contrary, Hristov et al[96] found reduced numbers of circulating EPCs in CHD patients on long-term statin treatment.

Statins appear to have a dose dependant effect on EPC count. A double blinded randomised pilot study found greater number of circulating CD34⁺ VEGFR-2⁺ EPCs after 12 wk of therapy with pravastatin 20 mg when compared to atorvastatin 10 mg[97]. Similarly, in ACS patients’ intensive statin therapy with atorvastatin 80 mg after primary or rescue PCI was associated with greater EPC count at 4-mo follow up as compared to 20 mg atorvastatin. The authors found no beneficial effect in an improvement of LV function[98]. Furthermore statin reloading in patients on moderate statin therapy undergoing percutaneous coronary intervention has been shown to increase EPC count[99,100] this correlates with the beneficial effect of statin reloading of high dose statin in patients on chronic therapy[80] mentioned above.

Several proposed intracellular signaling mechanisms accounting for the pleiotropic effect of statin therapy have been put forward. Figure 1 below summarizes the positive and negative effects on EPC proliferation, mobilisation and longevity but also the effect of statin therapy.

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Figure 1 Simplified diagram illustrating the positive and negative effects on endothelial progenitor cell proliferation, mobilisation and longevity together with proposed mechanisms of action of statin therapy. EPC: Endothelial progenitor cell; NO: Nitric oxide; eNOS: Endothelial nitric oxide synthase; VEGF: Vascular endothelial growth factor; mRNA: Messenger ribonucleic acid; TNF: Tumor necrosis factor alpha; IL-1: Interleukin 1; P13k-AKT: Phosphoinositide 3-kinase - protein kinase B pathway; NAD(P)H oxidase: Nicotinamide adenine dinucleotide phosphate-oxidase; miR : Micro non-coding ribonucleic acid.

EPCs have a pivotal role in the maintenance of vascular integrity. However, factors that influence EPC number, migration and function are now becoming recognised and have potentially a significant role in management of ischaemic heart disease patients. Statins once thought to modify cardiovascular risk only by lowering LDL-cholesterol are now being acknowledged as having alternative mechanisms that appear to have beneficial pleiotropic effects. One such mechanism may be mediated by EPCs. A number of studies have shown positive pleiotropic effect of statins on EPCs, both function and number. There appears to be a complex interaction between statins and EPC that is only now becoming recognised. Despite great progress since Asahara’s pioneering work, there remain gaps within our knowledge regarding the pleiotropic effect(s) of statins on EPCs. Further studies are required to elucidate and fully understand any pleiotropic effect and this may guide future beneficial therapeutic interventions.