Emerging role of PKA/eNOS pathway in therapeutic angiogenesis for ischaemic tissue diseases

Abstract

Although an abundant amount of research has been devoted to the study of angiogenesis, its precise mechanisms are incompletely understood. Numerous clinical trials focused on therapeutic angiogenesis for the treatment of tissue ischaemia have not been as successful as those of preclinical studies. Thus, additional studies are needed to better understand critical molecular mechanisms regulating ischaemic neovascularization to identify novel therapeutic agents. Nitric oxide (NO) plays a central role in ischaemic neovascularization through the generation of cyclic guanosine monophosphate (cGMP) and the activation of several other signalling responses. Accumulated evidence suggests that endothelial protein kinase A/endothelial NO synthase (PKA/eNOS) signalling may play an important role in ischaemic disorders by promoting neovascularization. This review highlights recent advances in the role of the PKA/eNOS and NO-cGMP-kinase cascade pathway in ischaemic neovascularization. We also discuss molecular relationships of PKA/eNOS with other angiogenic pathways and explore the possibility of activation of the NO/nitrite endocrine system as potential therapeutic targets for ischaemic angiogenesis.

1. Introduction

Ischaemic vascular diseases remain a leading cause of mortality and morbidity worldwide. Despite significant advances in medical and surgical intervention, the burden of these illnesses remains high. Restoration of blood flow to ischaemic organs is vital to prevent tissue death after arterial occlusion. As a strategy for the treatment of acute and chronic ischaemia, therapeutic angiogenesis evolved soon after the discovery of angiogenic growth factors.^1–3 Experimental studies showed initial promise of therapeutic angiogenesis in small animal and selected large animal models.⁴ However, after more than a decade of clinical investigation, none of the larger double-blinded, placebo-controlled trials of angiogenesis therapy in patients with various vascular diseases have demonstrated significant benefits.^5–8 Although the reason for this is complex, the lack of a comprehensive knowledge of biochemical and molecular mechanisms driving angiogenesis is a major obstacle to the development of effective therapeutic approaches.^9,10

Ischaemic neovascularization involves three major processes: angiogenesis, arteriogenesis (collateralization), as well as post-natal vasculogenesis, although the discrete importance of each process remains unclear.^11–13 Angiogenesis, the process of new capillary growth through sprouting from existing small blood vessels, is initiated from the activation of endothelial cells (ECs), followed by proliferation, migration, and tube formation. Ischaemia/hypoxia induces a variety of angiogenic factors that drive the formation of new vessels.¹² Arteriogenesis, including collateralization, refers to the outward growth and remodelling of existing arterioles into larger arteries when a main artery is obstructed. Among the various factors, mechanical forces including circumferential wall tension and shear stress seem to be primarily important for arteriogenesis.^14,15 Post-natal vasculogenesis is a process by which blood vessels are established de novo from endothelial progenitor cells (EPCs) derived from bone marrow (BM).¹⁶ These processes are collectively involved in regulating tissue neovascularization to restore blood perfusion back to the ischaemic region with vasculogenesis playing a complementary role by incorporating EPCs into the endothelium of new vessels (angiogenesis) or the remodelling of arteries (arteriogenesis).^12,17

Mechanistically, ischaemic neovascularization is stimulated by several chemical, physical, and mechanical factors through distinct but partially overlapping cellular and molecular pathways.^11,12 Ischaemia/hypoxia and concurrent acidic pH induce the expression of a set of angiogenic genes including growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor,^1,12,18,19 as well as chemokines such as monocyte chemoattractant protein-1 (MCP-1) and stromal-derived factor 1α (SDF-1α),^19,20 which can drive post-natal vasculogenesis.²¹ Mechanical factors such as circumferential wall tension may initiate arteriogenesis via the induction of smooth muscle MCP-1 expression,¹⁵ and shear stress signalling involving endothelial adhesion molecule PECAM-1¹⁴ is critical for arteriogenesis. It is noteworthy that inflammatory pathways also participate during ischaemic neovascularization. Together, the stimulation of ischaemic vascular remodelling is dynamic requiring multiple experimental approaches to understand key cellular and molecular events of neovascularization.

Extensive efforts have been made to better understand various intracellular signalling and molecular cascades of ischaemic neovascularization. It is clear that distinct ischaemic factors trigger multiple intracellular signalling pathways that converge into several pathways where endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) is involved. eNOS catalyses the conversion of l-arginine to l-citrulline generating NO that is important for the angiogenic activity of several factors including VEGF.^22–25 Akt/protein kinase B is regarded as a central upstream signalling regulator leading to eNOS activation.⁹ However, experimental evidence indicates that protein kinase A (PKA) is also an important mediator in eNOS activation during ischaemic neovascularization. This is in contrast with controversial roles; PKA may serve during angiogenesis.^26–29 Here, we discuss recent experimental advances of reactive oxygen species (ROS)/NO bioavailability, PKA/eNOS signalling, and NO-mediated cellular responses in ischaemic neovascularization and explore potential therapeutic uses of these targets to modulate ischaemic angiogenesis.

2. Neovascularization and NO bioavailability

Angiogenesis, vasculogenesis, and arteriogenesis are three principle events regulating tissue repair and remodelling in acute and chronic vascular diseases as mentioned earlier.³⁰ However, these processes are driven by separate, yet partially overlapping, cellular, and molecular pathways. Experimental studies have identified that angiogenesis and vasculogenesis involve various angiogenic growth factors such as VEGF, fibroblast growth factor (FGF), angiopoietins, platelet-derived growth factors (PDGF), and signalling molecules (e.g. NO), transcription factors, e.g. hypoxia-inducible factor-1 (HIF-1), various integrins, ephrins, and progenitor cells to enhance new blood vessel formation.^12,30–32 On the other hand, mechanical stimuli including oscillatory and steady laminar shear stress, and circumferential and longitudinal wall tension stretch stress regulate cell adhesion molecules and mitogenic factors that stimulate collateral vessel remodelling.^12,30–33 The following sections discuss the effect of NO metabolism and bioavailability during the processes of neovascularization.

2.1. Angiogenesis and NO bioavailability

Endothelium-derived NO plays a pivotal role in the maintenance of vascular tone via triggering smooth muscle relaxation, as well as in other pathophysiological processes, e.g. angiogenic response, of the EC.³⁴ NO induces smooth muscle cell (SMC) relaxation via the cGMP-PKG pathway, inhibiting calcium release through the IP3 receptor, decreasing intracellular calcium concentrations, and altering cytoskeleton organization.³⁵ During ischaemic conditions, NO modulates angiogenesis activity^24,36 through direct interaction of NO via S-nitrosylation of various proteins, including HIF-1.³⁷ Notably, NO production contributes to the angiogenic properties of VEGF in human ECs.³⁸ Evidences suggest that VEGF stimulates the release of NO from cultured human umbilical vein EC (HUVECs) by augmenting eNOS expression and phosphorylation.^39,40 Similar effects have been suggested for other factors, e.g. bFGF and substance P.^41,42 Moreover, VEGF- and FGF-induced angiogenesis is blunted by an NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME) in a rabbit cornea model of angiogenesis.^38,43,44 Likewise, angiogenesis activity can be diminished when NO bioavailability is reduced as evidence indicates that in vitro angiogenic activity is inhibited by oxidized LDL, which is known to decrease NO bioavailability.⁴⁵ Importantly, endothelium-dependent NO-mediated vasodilation and angiogenic response to hind limb ischaemia is blunted in hypercholesterolaemic rabbits.^45,46 Moreover, vascular remodelling during hind limb ischaemia is impaired in eNOS-knockout (KO) mice that cannot be reversed by exogenous VEGF therapy.⁴⁷

Precise mechanisms of NO-mediated angiogenesis are not completely understood, although studies support the hypothesis that NO stimulates both endothelial proliferation and migration.^47–49 The growth-promoting effect of NO is associated with cGMP generation in the cultured endothelium.^50,51 Importantly, NO along with cGMP production further reinforce this response by increasing VEGF and bFGF expression.^50,52,53 Studies have also shown that VEGF-induced angiogenesis is inhibited by an sGC inhibitor LY83583^39,43,48,50 and that cGMP production activates kinase cascades including PKG and mitogen-activated protein kinase (MAPK) cascades Ras, Raf1, and ERK.^50,54,55 NO enhances EC migration by stimulating EC podokinesis, enhancing the expression of αvβ3 integrin, and increasing bFGF-mediated dissolution of extracellular matrix.^47,53,56 Matrix metalloproteinase (MMP) degradation of matrix is important for vascular remodelling⁵⁷ and stimulates EC migration in part due to the activation of MMP-13 both in vitro and in vivo.⁵⁸ eNOS can also colocalize with membrane type 1-MMP on EC membrane and NO regulates compartmentation, activity, and function of MT1-MMP in ECs, making it an important target for NO regulation of EC migration, tube formation, and angiogenesis.⁵⁹ Conversely, it has been shown that eNOS gene transfer decreases MMP-2 and MMP-9 activities and inhibits smooth muscle migration.⁶⁰ Studies also suggest that NO-dependent peroxynitrite formation also regulates MMPs in different conditions such as in atherosclerotic plaque instability, myocardial injury, and tumour metastasis.^61–64 Therefore, although NO has diverse actions on MMPs, MMP-13 and MT1-MMP activities are required for NO-induced EC migration, tube formation, as well as angiogenesis. In summary, these and other cellular events are significantly influenced by NO production and bioavailability, thus modulating angiogenic activity involving EC proliferation, migration, and matrix remodelling (Figure 1).

Figure 1.

Prototypical angiogenic cytokine signalling pathways invoking endogenous NO production and subsequent kinase cascade activation. VEGF/VEGFR2 binding stimulates AKT/PKA mediated eNOS activation and NO production. NO mediated activation of sGC increases cGMP formation and PKG activity modulating subsequent MAPK cascades Ras, Raf, and ERK1/2 signalling. These events regulate EC proliferation and migration, and extracellular matrix remodelling resulting in angiogenesis.

2.2. Vasculogenesis and NO bioavailability

The BM-derived EPC is the key cell involved in vasculogenesis to restore blood flow in ischaemic tissue. Studies show that EPCs are mobilized from the BM into the systemic circulation, home to ischaemic sites, undergo in situ differentiation, and ultimately participate in the formation of new blood vessels.^16,65,66 This EPC mobilization cascade starts with peripheral ischaemia-induced tissue release of VEGF and subsequent activation of BM stromal NOS, resulting in increased BM NO production.^67,68 Besides this, the absolute number of EPCs in eNOS-KO mice and mice treated with l-NAME is significantly reduced, suggesting an important role of eNOS for basal EPC regulation.⁶⁹ Data also suggest that physical exercise also increases NO bioavailability and the number of EPCs, which is blunted by inhibition or deletion of eNOS.⁷⁰ Similarly, statin-induced NO-mediated EPC recruitment is blocked by treatment with l-NAME.⁷¹ Thus, NO bioavailability is also important for the stimulation of EPC mobilization and recruitment in the ischaemic site results in vasculogenesis.

2.3. Arteriogenesis and NO bioavailability

Although angiogenesis is ischaemia-driven, hypoxia is not required for arteriogenesis.⁷² As such, arteriogenesis activity occurs under normoxic environments far from ischaemic tissue regions.⁷³ Changes in mechanical forces acting on the ECs lining collateral vessels promote required inflammatory signals to start collateral remodelling.⁷⁴ As a form of vessel growth, pathological arteriogenesis may be related to increased shear and stretch forces as a consequence of increased flow through pre-existing collateral arterioles due to an increase in the pressure gradient following occlusion of a major artery.⁷⁵ NO derived from eNOS mediates vasodilation thus decreasing vascular resistance and also facilitates proportional vascular remodelling that controls changes in blood flow.⁷⁶ Arteriogenesis activity during ischaemia has been reported to involve a series of different factors including monocyte/macrophage-derived cytokines such as MCP-1, PECAM-1, and VEGF, as well as changes in shear stress of pre-existing collaterals that would activate eNOS acutely to stimulate arteriogenesis via outward vascular remodelling.^74,77 Yu et al.²⁵ showed that EC-derived NO plays a crucial role in arteriogenesis, pericyte recruitment, and stabilization of angiogenic vessels. NO also mediates the angiogenic effect by neuropeptide Y (NPY) in both hind limb ischaemia and chronic myocardial ischaemia models. NPY is evidenced to enhance capillary angiogenesis and collaterization via the activation of eNOS and up-regulation of angiogenic genes such as VEGF and PDGF.^78,79 Moreover, collateral vessel formation and subsequent recovery of blood flow were markedly increased in the ischaemic limb of eNOS transgenic mice.^80,81 In contrast, evidence from literature also demonstrates that NO prevents pro-inflammatory EC differentiation or expression of MCP-1.⁸² NO also inhibits SMC proliferation under certain vascular conditions.⁸³ Despite the above findings, the role of NO during collateral remodelling and arteriogenesis remains poorly understood.

3. Relationship of NO and ROS, and their effects on neovascularization

Proper functioning of the endothelium is often linked to the production and bioavailability of NO and regulation of ROS. Both ECs and vascular SMCs are capable of producing ROS from a variety of enzymatic sources. Studies have shown that ROS is produced in different subcellular compartments. NADPH oxidase and eNOS (uncoupled) are cell membrane-bound ROS-generating enzymes.^84,85 Mitochondria are also another major source of ROS production with primary locations of O₂^·− production at complexes I and III of the respiratory chain.⁸⁶ Cytosol and peroxisomes involve NADPH oxidase, eNOS, and xanthine oxidase to produce ROS. NADPH oxidase can also be located in the sarcoplasmic reticulum and produce ROS.^87–89 Free radicals have one or more unpaired electrons in their outer orbital, which make them highly unstable and prone to reaction with adjacent molecules by donating, abstracting, or even sharing their outer orbital electron. This can also result in the formation of secondary reactive species that can modify additional targets. An example of this is the reaction of NO with superoxide to form peroxynitrite that is a potent oxidant and nitrating molecule.⁹⁰ Critical detoxification mechanisms exist to regulate the formation of reactive species such as superoxide dismutases (SOD's), and glutathione peroxidases in conjunction with glutathione, thioredoxin, and peroxiredoxins facilitate reactive molecule decomposition to diminish intracellular redox imbalance.

Therefore, it is important that redox reactions having a short half-life be considered during studies of free radical/oxidant mechanisms of vascular disease and therapeutics. ROS can positively or negatively regulate vascular growth and remodelling. Although the exact nature of this relationship remains unknown, physiological or low levels of ROS are required for vascular growth. However, high amounts of ROS may react with various cellular components including the lipid, protein, and DNA resulting in loss of cell integrity, enzyme function, and genomic stability.⁹¹ Physiological levels of ROS from different sources under physiological conditions act as intracellular second messengers modulating proangiogenic signalling pathways affecting EC proliferation, EC barrier function, vasorelaxation, vascular remodelling, and VEGF-A signalling as well as post-natal vasculogenesis.^92,93 NADPH oxidase has been shown to play a role in the regulation of angiogenic growth factors such as VEGF and VEGF-induced angiogenesis in otherwise young, healthy animals.⁹⁴ Tojo et al.⁹⁵ further showed that ischaemic hind limb neovascularization is significantly impaired in gp91 phox-KO mice, possibly due to a reduction in production from inflammatory and local vascular cells. Hypoxia-induced production of NADPH oxidase can be inhibited with pharmacological inhibitors resulting in diminished ROS formation, decreased VEGF production, blunted Akt and ERK1/2 activation, and subsequently reduced angiogenesis.^96,97 It is also reported that ROS controls the VEGF expression by the activation of AP-1 transcription factor through subunit junB.⁹⁸ ROS overproduction in mitochondria has been also linked to a pro-angiogenic cellular status. Inhibiting ROS production by mitochondria complex 1 using rotenone was found to inhibit the expression of VEGF, in a tumour model, stopping angiogenesis in vivo and in vitro.⁹⁷ Conversely, excessive production of vascular superoxide contributes to impairment of endothelium-dependent vasorelaxation due to decreased NO bioavailability. Increased levels of peroxynitrite reduce tetrahydrobiopterin (BH₄) production and also a reduction in the BH₄-producing enzyme, guanosine triphosphate cyclohydrolase expression, contributing to eNOS uncoupling.^99,100 Specifically, peroxynitrite rapidly oxidizes active BH₄ to inactive dihydrobiopterin (BH₂), further leading to eNOS uncoupling.¹⁰¹ Moreover, peroxynitrite causes eNOS uncoupling through the disruption of zinc–thiolate cluster of eNOS by releasing zinc from the cluster and forming disulfide bonds between the monomers.¹⁰² Increased oxidation of BH₄ may be responsible for some of the cellular pathophysiological effects of enhanced ROS formation in the endothelium during vascular disease.^101,103 However, the precise amount and species specificity of ROS involved during EC dysfunction in vivo are not completely understood nor is it clear what concentration of ROS impairs neovascularization by inducing endothelial dysfunction and apoptosis resulting from reduced NO bioavailability.^92,93

Reduced NO bioavailability is unable to redirect blood flow through pre-existing collaterals due to impaired vasodilation, further reducing shear-mediated remodelling of collaterals thus reducing tissue blood flow. This reduction in blood flow reduces shear stress-dependent eNOS activation and subsequent NO release retarding further growth of collaterals, flow-mediated angiogenesis, and remodelling of angiogenic sprouting in the lower limb.^80,81 Although inflammatory signals may be needed for the start of the collateral growth, chronic inflammation is detrimental to vascular remodelling.⁷⁴ Excess amount of ROS generated during inflammation or ischaemic response may inhibit vascular growth and remodelling by inducing endothelial dysfunction including impaired vasomotor function, the recruitment of monocytes, diminished endothelial barrier function, and enhanced thrombosis.^104,105 In pathological conditions such as diabetes, atherosclerosis, hypertension, cardiac failure, and ischaemia reperfusion injury, excessive ROS generation or decreased NO bioavailability mediates EC dysfunction, cell proliferation, migration, inflammation, extracellular matrix deposition, apoptosis, fibrosis, cardiovascular growth, and remodelling.^106,107 From the above discussion, it is clear that increased ROS and subsequent reduced NO bioavailability impair vascular growth and remodelling, which highlights important targets for therapeutic angiogenesis in ischaemic myocardial infarction, peripheral vascular disease, as well as stroke. Significant advances have been made with respect to quantitative analytical methods to detect specific ROS and NO species that must be employed in future studies to better understand the relationship between ROS levels and stimulation of vascular remodelling in conjunction with NO bioavailability.

4. ROS, diabetes, and atherosclerosis

Studies indicate that eNOS function is impaired in diabetes and atherosclerosis as a result of increased vascular generation of ROS and reduced NO bioavailability in diabetic and hypercholesterolaemia patients and animal models.^108–110 Hypercholesterolaemia, hyperglycaemia, oxidized atherogenic lipoproteins, and advanced glycation end products are all linked to altered oxidative stress that can augment their production. High levels of glucose and lipid increase NADPH oxidase activity and impair the activities of mitochondrial complex enzymes leading to the formation of excessive ROS during diabetes and atherosclerosis.¹⁰⁷ Human atherosclerotic samples show a greater mitochondrial DNA damage than non-atherosclerotic samples highlighting increased ROS production in atherosclerosis. Moreover, mitochondrial damage precedes the development of atherosclerosis in apolipoprotein E-KO mice with or without heterozygous deficiency of SOD.^111,112 Thus, overproduction of ROS is associated with the activation of a variety of pro-inflammatory signals and inactivates anti-atherogenic enzymes such as eNOS and prostacycline synthase,¹¹³ resulting in decreased NO bioavailability in disease states. Therefore, excessive ROS production impairs ischaemic vascular remodelling and blockade of NADPH oxidase activity or scavenging of ROS restores post-ischaemic neovascularization in diabetes and atherosclerosis.¹¹⁴ ROS also impairs ischaemic neovascularization in hypercholesterolaemic animal models or human patients.^114,115 In these reports, the authors showed that both the macrovascular (blood flow recovery) and microvascular (capillary density) levels of ischaemia-induced neovascularization were significantly impaired with high cholesterol diet-induced hypercholesterolaemic mice with hind limb ischaemia. NADPH oxidase expression and oxidative stress was increased, but eNOS activity was decreased in ischaemic tissues of these hypercholesterolaemic mice.¹¹⁴ In contrast, NADPH oxidase expression, oxidative stress, and eNOS activity were not affected in NADPH oxidase-deficient hypercholesterolaemic mice that displayed augmented ischaemia-induced neovascularization during hypercholesterolaemia. Therefore, NADPH oxidase deficiency can prevent anti-angiogenic activity in atherosclerotic vessel disease through a reduction in ROS production and increasing NO bioavailability.¹¹⁴

It has been shown that diabetes-induced overproduction of ROS also impairs post-ischaemic neovascularization.¹¹⁶ As such, the inhibition of oxidative stress during experimental diabetes restores important mediators necessary for angiogenesis, such as VEGF-A signalling and ischaemic vasculogenesis responses. In addition, hyperglycaemia-induced overproduction of ROS also impairs EPC function leading to the impairment of angiogenesis and vasculogenesis by augmenting p38MAPK phosphorylation in bone marrow mononuclear cells (BM-MNCs) and reducing BM-MNC differentiation into EPCs in diabetes.^117,118 Treatment of diabetic animals with N-acetyl cysteine over time improves EC function, which may help to normalize impaired angiogenesis in diabetes.¹¹⁹ There is other evidence that pre-treatment of the diabetic rat aorta with SOD and/or antioxidant probucol prevents the impairment of endothelium-dependent relaxation in aortic rings.¹²⁰ Likewise, pre-treatment with either SOD or catalase has been shown to improve endothelial dysfunction in streptozotocin-induced diabetic rats, suggesting that vascular production of both superoxide and hydrogen peroxide is pathologically important.¹²¹ This is consistent with the observation that vasodilation, a mediator of arteriogenesis, is attenuated through an ROS reduction in NO bioavailability during diabetes.¹²² Likewise, collateral vessel formation, capillary density, and blood flow recovery are impaired in SOD-deficient mice due to production of excessive superoxide, decreased NO bioactivity, and increased apoptosis.¹²³ These findings demonstrate that vascular growth and remodelling is tightly balanced by both ROS-producing oxidases and ROS-scavenging enzymes particularly in disease states.

5. Signalling pathways leading to NO generation during ischaemic neovascularization

NO generation is now known to occur through multiple mechanisms including enzymatic and non-enzymatic pathways. Classical enzymatic mechanisms involve NO syntheses including eNOS, inducible nitric oxide synthase, and neuronal nitric oxide synthase that produce NO from l-arginine oxidation and reduction.¹²⁴ Other pathways such as the nitrite/NO system also generate NO via other non-classical pathways, through a reduction in nitrite anion back to NO.¹²⁴ Therefore, activation and subsequent interactions of various NO production pathways can be complex during chronic or intermittent tissue ischaemia.

5.1. Activation of the PKA/eNOS signalling pathway

Much effort has been made in defining the signalling pathways leading to the activation of eNOS. Regulation of eNOS activity is complex, involving various protein–protein interactions and numerous post-translational regulations such as serine/threonine phosphorylation at multiple sites.^125–127 Among them, the phosphorylation of serine residue at 1177 (Ser1177), which leads to maximal activation of eNOS activity, appears to be the most crucial and thus has been studied extensively. A number of kinases have been identified to phosphorylate eNOS, among them Akt-induced eNOS phosphorylation has been more clearly characterized.¹²⁷ Akt phosphorylates eNOS at Ser1177 and is involved in the regulation of basal activation of eNOS and agonist-mediated stimulation.¹²⁸ Akt is predominantly found in the cytosol in inactive form and must translocate to the membrane as a prerequisite to both its own activation via phosphoinositide-3-kinase (PI3K) and the phosphorylation of eNOS.¹²⁹ In addition to Akt, AMP-activated protein kinase (AMPK), protein kinase C (PKC), and PKA have been also shown to phosphorylate eNOS.¹²⁷ Initially, a biochemical study found that PKA phosphorylated eNOS at least on Ser633 and Ser1177.¹³⁰ Subsequently, eNOS phosphorylation at both sites was induced by shear stress in a PKA-dependent but Akt-independent manner in bovine aortic ECs (BAEC).^131–133 Compared with Akt, PKA induces eNOS activation in a two-phased pattern, which mainly involves Ser1177 and Ser617. The onset of phosphorylation on Ser1177 is quick and calcium-dependent, while Ser633 phosphorylation is slow and calcium-independent, suggesting that Ser633 phosphorylation maintains sustained high activity of eNOS after initial activation by calcium influx and Ser1177 phosphorylation.¹²⁷ However, compared with the well-recognized role of Akt in eNOS activation, less attention has been paid to the role of PKA in eNOS/NO-mediated function.

5.2. PKA/eNOS signalling pathway in neovascularization

To date, a growing list of stimuli has been identified that influence eNOS activation and NO synthesis by the activation of PKA, and many of them are activators of neovascularization^134–140 including vasoactive substances such as bradykinin and prostacyclin, and growth factors such as VEGF.^27,141 Bradykinin is a potent agonist for endothelial NO production through the PKA-dependent phosphorylation of eNOS on Ser1177.¹³⁸ A previous study showed that the bradykinin B2 receptor mediates a pro-angiogenic signalling.¹³⁹ Prostacyclin, the major product of cyclooxygenase in the macrovascular endothelium, acts on its endothelial receptors to promote NO production via the activation of the cAMP/PKA signalling pathway.¹⁴⁰ It is known that prostacyclin and NO act synergistically in VEGF-mediated angiogenic signalling.¹³⁴ Prostaglandin E2, another product of the cyclooxygenase pathway, has also been implicated in modulating angiogenesis via the PKA/eNOS pathway.¹³⁵ The role of the PKA/eNOS pathway in neovascularization has been documented in numerous well-controlled studies with pharmacological compounds such as forskolin^28,142 and cilostazol, cAMP inducers.^137,143 For example, Venkatesh et al.¹⁴⁴ reported that dipyridamole rapidly restores ischaemic limb blood flow and stimulates collateral artery perfusion via arteriogenesis activity through a PKA/eNOS/NO-dependent pathway. Similarly, Lu et al. have reported mechanisms of angiogenesis in Gab1-deficient mice revealing that the PKA/eNOS signalling pathway is critical in Gab1-mediated ischaemia and VEGF-induced angiogenesis both in vivo and in vitro. These studies also showed that Gab1 deficiency caused defective endothelial tube formation that could be rescued by re-introduction of either constitutively active PKA or eNOS.¹⁴¹

Apart from above-mentioned evidence, ischaemia/hypoxia is known to induce EPC homing,⁶⁷ which is of vital importance in vascular repair and vasculogenesis. As a target gene of HIFs, VEGF, which is an activator of the PKA/eNOS pathway, is critical for SDF-1α-mediated EPC homing.¹⁴⁵ Thus, it is possible that PKA/eNOS signalling may enhance EPC recruitment to the sites of ischaemia to promote vasculogenesis. Consistent with this idea, cilostazol stimulates progenitor cell integrin expression, migration, and adhesion through cooperative activation of PKA and Epac signals.¹⁴⁶ cAMP/PKA also enhances EPC differentiation via VEGFR2 and Neuropillin-1 induction as a blockade of PKA perturbs EPC differentiation and vascular formation in vitro and ex vivo, while over-expression of constitutive active PKA potently induces endothelial differentiation and vascular formation.¹⁴⁷

As discussed above, occlusion of a major artery increases mechanical stress through pre-existing collateral arterioles to facilitate vascular remodelling. Notably, mechanical forces are critical regulators of eNOS activity at both the transcriptional level and the post-translational level. Recent research suggests that laminar rather than oscillatory shear stress is the major activator of eNOS activity, and cyclic stretch seems to act as a secondary synergistic factor.^148,149 The mechanism of eNOS activation elicited by shear stress was first reported to be PKA but not Akt-dependent in BAEC. Further research confirms that shear stress induces a complex formed by Gab1/shp2/PKA/eNOS independent of Akt activation. Additionally, a two-phased eNOS activation pattern was observed when ECs were exposed to cyclic strain.¹⁵⁰ The first phase involves rapid phosphorylation of Ser1177 induced by calcium influx, and the second phase is sustained phosphorylation mainly through the PI3K–Akt signalling pathway. Thus far, it has been assumed that the activation of the eNOS/NO pathway mediates mechanical stress-induced angiogenesis through an activating PKA/eNOS pathway,^149,151 although a detailed intracellular signalling cascade needs to be further defined.

5.3. Cellular basis of PKA/eNOS-mediated neovascularization

Given the effects that the PKA/eNOS pathway exerts on all the processes of neovascularization, their mechanisms likely involve numerous targets at the cellular level. Studies revealed that the PKA/eNOS pathway regulates different processes through coordinated and synergistic modulation of multiple cell functions, such as endothelial proliferation, tube formation, and survival; induction of relaxation and cell arrest of vascular SMC;^152,153 and stimulation of homing and differentiation of EPCs. In ECs, PKA/eNOS signalling favours endothelial proliferation, tube formation, and survival partially through the crosstalk with the PI3K/Akt pathway, activating cell cycle regulators and phosphorylating the anti-apoptotic Bcl-2 family members.^154,155 In addition, the PKA/eNOS pathway induces endothelial migration via NO production and calcium influx by activating ion channels¹⁵⁶ and promotes endothelial wound healing through cytoskeletal reorganization of focal adhesions.¹³⁵ Conversely, pre-treatment with PKA or eNOS inhibitors both significantly attenuates cilostazol-induced endothelial tube formation.¹³⁷ At the subcellular level, recent studies indicate that PKA and eNOS are co-localized in the endothelial lamellipodia located at cell contacts, indicating a possible role in regulating cell junction and vascular permeability.¹⁵⁷ Indeed, PKA and Epac were demonstrated to stabilize EC junctions and enhance cell barrier function via integrin-dependent and -independent pathways.¹⁵⁸ Moreover, NO derived from pericytes plays an important anti-inflammatory role in mediating endothelial response to acute infection.^152,153 Endothelium-derived NO triggers the relaxation of vascular SMCs¹⁵⁹ and can inhibit SMC proliferation.⁸³ However, the importance of these responses for artery remodelling and arteriogenesis in ischaemic tissues is not well understood.¹⁶⁰ Additionally, NO also regulates EPC survival, migration, and homing. SDF-1α inhibits EPC apoptosis mainly through the Akt/eNOS pathway,¹⁶¹ and eNOS/NO nitrosylation of matrix metallopeptidase 9 (MMP-9) is critical for EPC mobilization and migration.¹⁶² However, future studies will be needed to explore the relationship and importance of PKA/eNOS signalling for these responses under ischaemic settings.

5.4. NO/nitrite/nitrate system

The NO metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) anions were traditionally thought to be inert end products of endogenous NO oxidation. However, recent studies show that these anions can be recycled in vivo to form NO and other bioactive nitrogen oxides representing an important alternative source of NO to the classical l-arginine/NO synthase pathway particularly in hypoxic states.¹⁶³ Bioactivation of nitrate to nitrite from dietary or endogenous sources requires its entry into the enterosalivary system whereby nitrate is reduced to nitrite by oral commensal bacteria. However, nitrite is unique in its redox position between oxidative and reductive chemistry and its relative stability in blood and tissues.^164,165 Nitrite can be reduced to NO by numerous pathways such as deoxyhaemoglobin, deoxymyoglobin, xanthine oxidoreductase, ascorbate, polyphenols, and protons.¹⁶³ A reduction in nitrite to NO is increased under ischaemic conditions particularly when oxygen-dependent NOS activities are compromised. Thus, bioavailable NO equivalents can be stored in blood and tissue in the form of nitrite, which can act in an endocrine fashion within the blood, accumulate in various tissues, and undergo reduction back to NO under certain pathophysiological states.^124,166 Together, the nitrite/NO system works in cooperation to maintain NO bioavailability in NOS-dependent and -independent ways in conjunction with blood and tissue factors.

We have shown that the nitrite/NO system can be activated in a PKA-dependent manner to stimulate arteriogenesis and angiogenesis, thus restoring blood flow in ischaemic tissue.¹⁴⁴ Similarly, other studies have shown that adenosine-dependent eNOS activation via adenylate cyclase and PKA activation leads to tissue production of NO implicating initiation of nitrite/NO endocrine responses and their interaction with other pathways.^167,168 This is further supported by Elrod et al.¹⁶⁹ that reported cardiac-specific eNOS transgenic-derived NO transported via plasma nitrite acts in distant organs to attenuate cellular injury during ischaemia. Finally, reports also reveal that nitrite reduction to NO and NO-modified proteins during pathological and physiological hypoxia contributes to physiological hypoxic signalling, vasodilation, modulation of cellular respiration, and the cellular response to ischaemic stress.^163,170 Thus, the NO/nitrite system is an important endocrine pathway to maintain NO particularly in hypoxic conditions that is regulated by PKA.

5.5. Signalling interactions between PKA/eNOS and other angiogenic pathways

As a recently defined angiogenic regulator, the PKA/eNOS pathway interacts with many conventional angiogenic-signalling pathways. Among these, some serve as substrates of PKA, others act in parallel with PKA, while converging synergistically at eNOS activation. Epac is the other major ubiquitously expressed target of cAMP aside from PKA, and it often acts synergistically with PKA in regulating endothelial behaviours such as tube formation, migration, and barrier function. Forskolin, a cAMP inducer, stimulates in vitro HUVEC tube formation and in vivo angiogenesis through a coordinated crosstalk between PKA-dependent CRE-mediated VEGF expression and Epac-dependent Erk activation and PI3K/Akt/eNOS signalling.¹⁴² The activation of Epac1 and PKA by cAMP results in the stimulation of two parallel signalling pathways compensating each other, enhancing endothelial integrity and barrier function by modulating circumferential actin, while promoting HUVEC integrin-mediated migration.¹⁷¹ The PI3K/Akt pathway acts synergistically with the PKA/eNOS pathway to modulate endothelial angiogenesis. Boo et al. suggested that coordinated interaction between Akt and PKA is an important mechanism in regulating eNOS activity in response to mechanical shear stress.¹³¹ Besides, cilostazol enhances tube formation in human aortic ECs by inducing NO production via a cAMP/PKA- and PI3K/Akt -dependent eNOS activating mechanism.¹³⁷ Finally, cAMP/PKA signalling can activate p85α subunit of PI3K and trigger Akt activation, inhibiting endothelial apoptosis, while maintaining proliferation, thus exerting a protective effect against vascular injury.¹⁷²

Apart from those mentioned above, the PKA/eNOS pathway also interacts with AMPK/eNOS and PLC/PKC/eNOS pathways. While PLC/PKC activation inhibits eNOS activation via Thr495 phosphorylation and Ser1177 de-phosphorylation on eNOS,¹⁷³ AMPK activation enhances eNOS activity.^174,175 cAMP-elevating agents significantly attenuate AMPK Thr172 phosphorylation and enhance Ser485/491 phosphorylation through PKA activation in several different cell lines, attenuating its activity.¹⁷⁴ Thrombin and histamine induce AMPK phosphorylation on Thr172 and eNOS phosphorylation on Ser1177, and the latter is strongly inhibited by pre-treatment of H-89, an inhibitor for PKA and AMPK, although was unaffected by the cAMP competitive analogue cAMPs, indicating the involvement of both signalling pathways.¹⁷⁵ Furthermore, in an in vivo rat hind limb ischaemia model, a calcitonin gene-related peptide promotes blood flow recovery and angiogenesis through activating eNOS phosphorylation on Ser1177 via AMPK in HUVEC, which was blocked by a cAMP/PKA inhibitor.¹³⁶

In summary, all the evidence suggests that under pathophysiological conditions, various stimuli are able to promote neovascularization involving the PKA/eNOS signalling pathway. PKA signalling interacts with other pathways such as Akt and AMPK, converges on the activation of eNOS, and leads to vessel vascular growth (Figure 2). Given the complex nature of PKA signalling, it remains a salient challenge to determine the coordination of multiple signalling interactions that may finely modulate neovascularization. In addition, these interactions were largely examined with in vitro cultured cell system and in vivo ischaemic neovascularization is complex with multiple factors (mechanical, chemical, metabolic, and immunological), influencing the process of tissue injury and repair. Thus, it is perhaps not surprising that even more complicated interactions of signalling pathways are involved in ischaemic neovascularization.

Figure 2.

PKA-dependent signalling pathway is activated by various stimuli (e.g. mechanical forces and chemical ligands) regulating vessel growth. PKA signalling interacts with other intracellular pathways such as Akt and AMPK, converging onto the activation of eNOS that exerts multiple cellular functions synergistically leading to vessel growth. It should be pointed out that the actions of eNOS/NO on the phenotypes of ECs and SMCs may be context-specific dependent on several physiological and pathological conditions.

6. Different therapies that potentiate the PKA/eNOS and nitrite/NO system

There are some drugs that have shown their effects involving PKA/eNOS and nitrite/NO signalling pathways. Among them, we discuss two that have clearly been reported to influence these pathways including statins and dipyridamole.

6.1. Statins

Statins are established anti-hypercholesterolaemia drugs that can stimulate ischaemic angiogenesis through an Akt/eNOS/NO-dependent pathway.¹⁷⁶ Brouet et al.¹⁷⁷ also found that statins stimulate hsp90 and Akt/eNOS pathway which helps prevent eNOS uncoupling, thus maintaining eNOS activity and NO bioavailability. Harris et al. further showed that the treatment of BAEC with statins stimulated eNOS phosphorylation at Ser1179 and Ser617, which was blocked by the PI3 kinase inhibitor wortmannin, and phosphorylation at Ser635 was blocked by the PKA inhibitor KT-5720. Thus, demonstrating that eNOS is acutely activated by statins through both PKA and Akt.¹⁷⁸ Similarly, statins improve EPC mobilization and myocardial neovascularization after infarction by increasing endothelial NO bioavailability. Statins enhance endothelial NO bioavailability by both promoting endothelial production and preventing NO inactivation by free radicals.^176,179,180 Finally, it has also been reported that statin therapy stimulates neovascularization at the myocardial infarct border zone of mice and this effect is absent in eNOS-KO mice.¹⁸¹ In summary, statins induce ischaemic neovascularization via both PKA/eNOS and Akt/eNOS pathways but have not been useful in stimulating clinical therapeutic angiogenesis.

6.2. Dipyridamole

As briefly mentioned earlier, we have recently examined the effect of dipyridamole on therapeutic angiogenesis.¹⁸² Dipyridamole augmented angiogenesis owing to PKA-dependent eNOS activation which enhanced the production of NO and subsequent plasma nitrite. Dipyridamole therapy not only increased eNOS activity but also increased NO bioavailability in tissues, leading to an increase in plasma nitrite anion in non-ischaemic tissue supporting the hypothesis of an endocrine nitrite/NO pathway that was regulated in a PKA-dependent manner.¹⁶⁹ Dipyridamole can also augment downstream signalling pathways of NO by increasing intracellular levels of cGMP, thereby potentiating NO/cGMP-mediated vasodilation and enhancing ischaemia-induced angiogenesis.¹⁸³ Thus, dipyridamole stimulates ischaemic neovascularization via an activating PKA–eNOS–NO–cGMP pathway.

While both pharmaceutical agents highlight the potential importance of PKA/eNOS signalling for vascular remodelling therapy, novel approaches targeting these pathways may also be useful.

7. Perspectives and conclusions

The concept of therapeutic angiogenesis remains an attractive medical approach for the treatment of ischaemic diseases. However, a better understanding of the complex molecular mechanisms and intracellular signalling network underlying ischaemic neovascularization is necessary to provide more effective strategies. Therefore, it is important to clarify interactions among the NO-related signalling pathways that can increase NO bioavailability in ischaemic neovascularization. It is well known that the bioavailability of NO is increased by eNOS phosphorylation either by Akt or PKA and that NO exerts its effect via sGC/cGMP kinase pathways. However, NO is an unstable gasotransmitter with a short half-life influenced by the presence of oxygen-derived free radicals. Moreover, it is difficult to selectively administer NO discretely to tissues in vivo. Thus, additional study is needed to develop novel pharmaceutical approaches to increase NO bioavailability involving PKA-dependent signalling. Additional molecular studies are necessary to determine the exact role of PKA in vascular remodelling response using sophisticated genetic models and specific pharmacological interventions. Due to the signalling complexity of PKA with many substrates and interacting proteins,^184,185 a combination of approaches involving proteomics and system biology may be useful in elucidating roles of PKA/eNOS in ischaemic neovascularization.

Conflict of interest: C.G.K. has intellectual property on the use of nitrite for ischaemic vascular remodelling and a financial interest in TheraVasc Inc.

Funding

C.G.K. is the recipient of an ADA Basic Science Grant 1-10-BS-84 and NIH Grant HL80482 and S.C.B is funded by a fellowship from the Malcolm Feist Cardiovascular Research Endowment, LSU Health Sciences Center, Shreveport. J.L. is supported by research grants from the National Science Funds of China (nos 81070115 and 81170098) and the Major State Basic Research Development Program of China (no. 2012CB945100).