PPARs and angiogenesis

The PPAR (peroxisome-proliferator-activated receptor) family consists of three ligand-activated nuclear receptors: PPARα, PPARβ/δ and PPARγ . These PPARs have important roles in the regulation of glucose and fatty acid metabolism, cell differentiation and immune function, but were also found to be expressed in endothelial cells in the late 1990s. The early endothelial focus of PPARs was PPARγ , the molecular target for the insulin-sensitizing thiazolidinedione/glitazone class of drugs. Activation of PPARγ was shown to inhibit angiogenesis in vitro and in models of retinopathy and cancer, whereas more recent data point to a critical role in the development of the vasculature in the placenta. Similarly, PPARα, the molecular target for the fibrate class of drugs, also has anti-angiogenic properties in experimental models. In contrast, unlike PPARα or PPARγ , activation of PPARβ/δ induces angiogenesis, in vitro and in vivo, and has been suggested to be a critical component of the angiogenic switch in pancreatic cancer. Moreover, PPARβ/δ is an exercise mimetic and appears to contribute to the angiogenic remodelling of cardiac and skeletal muscle induced by exercise. This evidence and the emerging mechanisms by which PPARs act in endothelial cells are discussed in more


PPAR ligands
Owing to their effects on metabolism, the primary therapeutic areas for pharmaceutical PPAR ligand development have been dyslipidaemia for PPARα and PPARβ/δ and Type 2 diabetes for PPARγ .The PPARs all appear to have a large flexible ligand-binding domain which allows them all to be activated by a large variety of fatty acids and eicosanoid mediators in vitro [9].It has, however, been difficult to assess which, if any, of these mediators are true endogenous ligands.The best defined endogenous PPAR ligand yet described is the fatty acid synthase product POPC (1-palmitoyl-2-oleoylsn-glycerol-3-phosphocholine) which is produced in the liver, and binds and selectively activates hepatic PPARα in vivo [10].Whether POPC acts as a universal PPARα and the true nature of ligands for the other PPARs have yet to be determined.
Because PPARα and PPARγ ligands independently are useful clinical drugs in the treatment of commonly coexpressed metabolic disorders, synthetic dual PPARα/γ or pan-PPAR ligands have also been developed and show a combined clinical efficacy [12], although independent safety concerns have led to a number of late-stage failures.
experimental studies using the clinically used PPARα agonist fenofibrate show that PPARα activation inhibits angiogenesis in vitro, in vascular remodelling and in tumour cell growth.Fenofibrate inhibits human dermal endothelial cell proliferation, migration and tube formation in vitro [13] via disruption of the cytoskeleton and inhibition of the critical endothelial cell survival factor Akt [13].Moreover, in a model of post-coronary artery angioplasty remodelling in the pig, post-angioplasty adventitial angiogenesis was also significantly reduced by fenofibrate treatment [14].
PPARα has been detected in the invading microvessels in human prostate cancer and in mice implanted with human pancreatic cancer cells [15].In vitro and in vivo, PPARα activation inhibited VEGF (vascular endothelial growth factor)-induced endothelial cell migration and fibroblast growth factor-2-induced corneal angiogenesis [15].Systemic PPARα ligand treatment of mice with implanted melanoma, glioblastoma and fibrosarcoma led to a reduction in tumour growth and a reduction in number of microvessels [15].To show that PPARα-mediated reduction in angiogenesis was critical in this process, transformed PPARα-null fibroblasts were implanted into wild-type and PPARα − / − mice.The growth of the transformed PPARα-null cells could only be inhibited by PPARα activation in wild-type, but not PPARα − / − mice [15].At the molecular level (Figure 1), PPARα activation in this model was associated with the production of the anti-angiogenic factors thrombospondin-1 and endostatin [15].
A common diabetic complication is retinopathy, which is driven by an inflammatory angiogenesis.Although clinical evidence for an anti-angiogenic role of PPARα is limited, there is now evidence for a protection against retinopathy in diabetic patients treated with fenofibrate.Although clearly not a major disrupter of vascular function, in the 'Fenofibrate Intervention in Event Lowering in Diabetes (FIELD)' study, fenofibrate demonstrated a significant 30 % reduction in the need for laser therapy in patients with or without known diabetic retinopathy [16].

PPARγ
Once PPARγ was discovered in endothelial cells, it was clear very early that PPARγ activation led to the inhibition of angiogenesis in vitro [7,8] and in vivo demonstrated using a corneal model of VEGF-induced angiogenesis [8].PPARγ activation decreases pro-angiogenic VEGF responses by suppressing transcription of its receptor VEGFR2 (Figure 1), by interacting with and preventing Sp1 (specificity protein 1) binding to DNA [17].Similar results have also been found in VEGF and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model [18], and in a laser photocoagulation-induced choroidal neovascularization in rats and cynomolgus monkeys [19].In ischaemia-induced retinopathy in mice, PPARγ ligand therapy is protective in a manner sensitive to the production of the protective adipokine adiponectin [20], whereas, in a similar mouse ischaemic retina model, the protective antiangiogenic effects of ω − 3-PUFA (polyunsaturated fatty acid) treatment were also shown to be mediated by PPARγ .At the molecular level, a PPARγ antagonist GW9662 reversed the ω − 3-PUFA-mediated reduction of retinal tumour necrosis factor-α, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin and angiopoietin 2, but not VEGF [21].Clinically, rosiglitazone may also delay the onset of proliferative diabetic retinopathy.In a longitudinal review of patients treated with rosiglitazone (rosiglitazone group, 14 eyes; control group, 24 eyes), progression to peripheral diabetic retinopathy over 3 years occurred in 19 % in the rosiglitazone group and 47 % in the control group, representing a 59 % relative risk reduction [22].In this study, the incidence of diabetic macular oedema was similar between control and rosiglitazone groups; however, it must be noted that a common and potentially important limiting side effect for PPARγ ligand therapy for diabetic retinopathy is a mild/moderate increase in the incidence of macular oedema [23].
PPARγ expression is associated with a vast number of cancers [24].PPARγ may also be of benefit in limiting cancer growth and spread by reducing angiogenesis.PPARγ is found at higher levels in proliferating endothelial cells, potentially making these cells more susceptible to ligand therapy [25].In vivo, the PPARγ ligand rosiglitazone inhibits angiogenesis in the chick chorioallantoic membrane and the cornea, and metastasis in glioblastoma, Lewis lung cell carcinoma, liposarcoma or rhabdomyosarcoma implanted in mice [25].
PPARγ also is critical in development in the mouse.PPARγ − / − mice exhibit early embryonic lethality due to severe alterations of the placental vasculature [26].These vasculature defects are associated with an imbalance of pro-and anti-angiogenic factors, with increased levels of proliferin and decreased levels of proliferin-related protein.Moreover, pregnant wild-type mice treated with rosiglitazone C The Authors Journal compilation C 2011 Biochemical Society showed an altered placental microvasculature, and a reduced expression of proliferin, VEGF and platelet-endothelial cell adhesion molecule-1 [27].Whether PPARγ is critical for human placental growth and development is not known.

Pro-angiogenic properties
Although the weight of evidence points to predominantly anti-angiogenic activities for PPARα and PPARγ , a number of studies do contradict these findings, and it is conceivable that local environmental factors may additionally govern how PPARs regulate angiogenesis at the local tissue level.Interestingly, the VEGF promoter does contain a PPARresponse element and PPARα and PPARγ ligands are capable of inducing VEGF [28][29][30][31] (Figure 1) and angiogenesis in an endothelial/interstitial cell co-culture assay in vitro and in a murine corneal angiogenesis model in vivo [28].The angiogenesis induced in this assay by PPARα and PPARγ ligands was associated with the induction of VEGF, increased activation of Akt and phosphorylation (activation) of eNOS (endothelial nitric oxide synthase) (NOS3) [28].These results are controversial, as previous corneal angiogenesis models clearly demonstrate anti-angiogenic effects of PPARα and PPARγ ligands [8,15,32].Interestingly, however, therapeutic angiogenesis has been demonstrated both in hind limb [33] and cerebral ischaemia [34] with PPARγ ligands, which was associated in both cases with eNOS activation.
Subcutaneous Lewis lung carcinoma and B16 melanoma implanted in PPARβ/δ − / − mice exhibit a diminished blood flow and immature microvascular structures, which can be rescued by re-expression of PPARβ/δ [37].Importantly, in pancreatic tumours removed from patients, PPARβ/δ expression strongly correlated with the advanced pathological tumour stage and increased risk of tumour recurrence and distant metastasis [41].PPARβ/δ has therefore been suggested as an important 'hub node' transcription factor, regulating the tumour angiogenic switch [41].
PPARβ/δ may also have an important role in physiological angiogenesis.Skeletal muscle-specific overexpression of constitutively active PPARβ/δ in mice leads to a dramatic increase in running endurance [42,43].Similarly, treatment with the highly selective PPARβ/δ agonists GW0742 and GW501516 give the heart an exercise phenotype.GW0742 and GW501516 both induced a surprisingly rapid (after 24 h) remodelling of mice hearts [44] and skeletal muscle [45], by increasing microvessels numbers and density.This remodelling in cardiac and skeletal muscle was identical with the phenotype observed with exercise and was mediated by the activation of calcineurin.Calcineurin activation leads to the activation of NFATc3 (nuclear factor of activated Tcells c3) and the induction of its pro-angiogenic target HIF-1 (hypoxia-inducible factor 1) [44].

Conclusions
The PPARs are a group of ligand-activated transcriptional regulators that sit at the intersection of genes and the dietary environment.PPARα and, in particular, PPARγ show predominantly anti-angiogenic properties in vitro and in animal models.PPARα and PPARγ are both activated by clinically used drugs: the fibrates and glitazones/thiazolidinediones.Clinically, these drugs have been used for a number of years and, although they do not appear to be primary potent angiostatic compounds, this does not rule out potentially beneficial actions on pathophysiological angiogenesis such as diabetic retinopathy or a variety of cancers, where the levels of PPARα or PPARγ may be elevated.This review has concentrated on direct effects of PPARs on the endothelial cell.However, PPARs are expressed in a variety of inflammatory and stromal cell types where whole-tissue PPAR activity may have an important influence on any angiogenic outcome.PPARβ/δ, unlike PPARα or PPARγ , appears to be a predominantly pro-angiogenic signalling molecule particularly in cancer and in the physiological muscle remodelling with exercise.One PPARβ/δ ligand GW501516 is in clinical trials for dyslipidaemia.It will be interesting to see how the PPARβ/δ field develops in this clinical setting as caution may be required when testing PPARβ/δ activation in aged populations where more pathological angiogenesis may play important pathological roles.

Figure 1
Figure 1 PPAR regulation of anti-and pro-angiogenic pathways