Ischemic Neoangiogenesis Enhanced by 2-Adrenergic Receptor Overexpression
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循环研究杂志 2005年第11期
Guido Iaccarino, Michele Ciccarelli, Daniela Sorriento, Gennaro Galasso, Alfonso Campanile, Gaetano Santulli, Ersilia Cipolletta, Vincenzo Cerullo, Vincenzo Cimini, Giovanna Giuseppina Altobelli, Federico Piscione, Ornella Priante, Lucio Pastore, Massimo Chiariello, Francesco Salvatore, Walter J. Koch, Bruno Trimarco
the Dipartimento di Medicina Clinica e Scienze Cardiovascolari ed Immunologiche (G.I., M.C., D.S., G.G., A.C., G.S., E.C., F.P., O.P., M.C., B.T.), Universite?Federico II, Napoli, Italy; Dipartimento di Biochimica e Biotecnologie Mediche (V.C., L.P., F.S.), Universite?Federico II, Napoli, Italy; CEINGE-Biotecnologie Avanzate (V.C., L.P., F.S.), Napoli, Italy; Dipartimento di Scienze Biomorfologiche e Funzionali (V.C., G.G.A.), Universite?Federico II, Napoli, Italy; Center for Translational Medicine (W.J.K.), Thomas Jefferson University, Philadelphia, Pa; and IRCCS Neuromed (B.T.), Pozzilli, Italy.
Abstract
2-Adrenergic receptors (2ARs) are widely expressed, although their physiological relevance in many tissues is not yet fully understood. In vascular endothelial cells, they regulate NO release and vessel tone. Here we provide novel evidence that 2ARs can regulate neoangiogenesis in response to chronic ischemia. We used in vivo adenoviral-mediated gene transfer of the human 2AR to the endothelium of the rat femoral artery and increased 2AR signaling resulting in ameliorated angiographic blood flow and hindlimb perfusion after chronic ischemia. Histological analysis confirmed that 2AR overexpression also produced benefits on capillary density. The same maneuver partially rescued impaired angiogenesis in spontaneously hypertensive rats (SHR), whereas gene delivery of the G-proteineCcoupling defective mutant Ile164 2AR failed to provide ameliorations. Stimulation of endogenous and overexpressed 2AR on endothelial cells in vitro was found to regulate cell number by inducing proliferation and [3H]-thymidine incorporation through means of extracellular receptor-activated kinase and vascular endothelial growth factor. The 2AR also has novel effects on endothelial cell number through stimulation of proapoptosis and antiapoptosis pathways involving p38 mitogen-activated protein kinase and PI3-kinase/Akt activation. Therefore, 2ARs play a critical role in endothelial cell proliferation and function including revascularization, suggesting a novel and physiologically relevant role in neoangiogenesis in response to ischemia.
Key Words: angiogenesis rats polymorphism hypertension in vivo digital angiography
Introduction
The endothelium controls several vascular functions, including vascular tone and permeability, thrombosis, hemostasis, and angiogenesis.1 It is noteworthy that all these functions can be regulated by activation of receptors, and often, the same receptor can activate multiple endothelial functions.
Adult angiogenesis only occurs in particular conditions such as wound healing, tumorogenesis, hypoxia, and chronic ischemia.2 It is a phenomenon intimately associated with endothelial cell (EC) proliferation, which appears to be under control of the extracellular receptor-activated kinase (ERK)/mitogen-activated protein kinase (MAPK)eCmediated signaling cascades.2 The most important system regulating angiogenesis is the cytokine vascular endothelial growth factor (VEGF), although a number of other cytokines and hormones acting through various tyrosine kinase and G-proteineCcoupled receptors are also implicated in this process.
The adrenergic system is the major regulator of cardiac and vascular function, and evidence is mounting for the relevance of this system in the control of endothelial vasodilation through means of 2- and -adrenergic receptors (ARs). In particular, 2ARs, the most abundant ARs in the vasculature,3,4 modulate the release of NO, causing endothelium-dependent vasodilation.5 2ARs are G-proteineCcoupled receptors activated by adrenergic catecholamines and promote a series of intracellular signal transduction pathways, leading to multiple cell-specific responses.6,7 Recently, it was proposed that 2ARs modulate cell proliferation, at least in fibroblasts, by activating ERK/MAPK through pathways dependent on -arrestins.8 The physiological implications of such an in vitro observation have not yet been fully investigated in vivo.
Altogether, the above considerations provide the background for the current investigation into the role of 2ARs in the control of angiogenesis. Accordingly, we evaluated whether 2ARs on ECs could enhance ischemia-induced angiogenesis in vivo. Also, in ECs, we evaluated the in vitro effects of 2AR overexpression on cell signaling and biology.
Materials and Methods
In Vitro and In Vivo Procedures
Experimental procedures were performed as described previously.5,9eC13 Extended details are provided in the online data supplement, available at http://circres.ahajournals.org.
Results
Effects of In Vivo 2AR Gene Transfer to the Endothelium During Chronic Ischemia in Wistar Kyoto Rats
We studied neoangiogenesis in a well-characterized rat model of chronic hindlimb ischemia in the rat.14 Our control group consisted of pooled data collected from sham-treated and AdEmpty viruseCtreated rats because no differences were found between these two treatments (supplemental Table I). In this model, 15 days after resection of the femoral artery, we observed a reduction in AR density within the ischemic hindlimb, which was attributable to a selective downregulation of 2ARs, the major AR subtype found in this tissue (Table). The numbers included in the Table reflect the sum of ARs expressed on all cell types present in the hindlimb area, including, but not solely, ECs. Also, 15 days after femoral artery resection, there was impairment in limb perfusion assessed by digital angiography (Figure 1A; supplemental video 1). These hemodynamic changes were paralleled by the rarefaction in capillary (<5 e蘭) density in the anterior tibial muscle compared with the contralateral normal limb (Figure 1C and 1D). In a subset of rats, we injected the Ad2AR-wt into the femoral artery at the time of surgery. Consistent with previous results,15 this resulted in 2AR overexpression in muscle, arterial capillaries, and venous (Figure 1E), which was double that of endogenous AR density found in the rat hindlimb (Table). After 15 days of ischemia, rats receiving Ad2AR-wt had significantly improved hindlimb perfusion with increased blood flow compared with untreated rats (Figure 1A and 1B; supplemental video 2). Similarly, histological analysis of the anterior tibial muscle revealed restored capillary density because of Ad2AR-wt treatment (Figure 1C and 1D). These positive benefits in vivo appear to be attributable to enhanced 2AR signaling because delivery of Ad2AR-Ile164 (Figure 1A through 1D) resulted in no beneficial effects on hindlimb hemodynamic or histology. The Ile1642AR mutation is a naturally occurring human polymorphism that causes a severe impairment in the G-protein coupling.16eC19
Chronic ischemia leads to increased systemic VEGF levels, a potent proangiogenesis cytokine, that typically return to baseline values once the ischemia is eliminated.20,21 We examined VEGF levels in our model in the blood and in the nonischemic contralateral hindlimb. In both samples, VEGF-165 levels were significantly lower in ischemic rats treated with Ad2AR-wt compared with control and Ad2AR-Ile164 rats, suggesting that 2AR overexpression significantly lessens ischemia (Figure 1F and 1G). Overall, these results suggest that 2ARs can regulate key endothelial cellular functions in vivo in response to chronic ischemia.
Impaired Angiogenesis Ameliorated by In Vivo 2AR Gene Transfer to the Endothelium During Chronic Ischemia in Spontaneously Hypertensive Rats
To further explore the role of endothelial 2AR in pathophysiological conditions, we repeated the in vivo experiment using the angiogenesis-impaired spontaneously hypertensive rat (SHR).22 Previously in this model, we have shown that reduced vascular and endothelial AR signaling and vasodilation is corrected by 2AR gene transfer.5 Fifteen days after resection of the femoral artery in SHR, there was a dramatic impairment of hindlimb perfusion, evidenced by the elevated occurrence of blistering, necrosis, or self-inflicted amputation of the ischemic paw (occurrence of necrosis, amputation, and blistering: SHR 35%; Wistar Kyoto [WKY] 0%; P<0.05; 2 test). This loss of perfusion was confirmed by digital angiography (Figure 1A), dyed microsphere dilution (Figure 1B), and by histological analysis (Figure 1D). In SHR treated with the AD2AR-wt, we observed a partial correction of this phenotype indicated by the reduced occurrence of blistering (10%; P<0.05; 2 test) and the ameliorated hemodynamic and histological parameters (Figure 1A, 1B, and 1D).
Finally, the analysis by Western blot of VEGF performed on the contralateral nonischemic hindlimb showed that systemic levels of this cytokine were reduced in the Ad2AR-wt SHR compared with the nontreated group, suggesting that local ischemia was resolved (Figure 1G).
Effects of 2AR Signaling on EC Proliferation
Our in vivo results do not let us discern whether the protective effects of 2AR overexpression in the rat hindlimb arterial endothelium after ischemic damage are attributable to an effect on cell proliferation or cell survival. These mechanistic questions were addressed in vitro using ECs in culture. The overall angiogenic properties of 2AR were recapitulated under conditions in which stimulated human ECs organize in network forming tubules. The AR agonist isoproterenol (ISO) increased the number of connections among endothelial tubules in vitro, and this response was magnified by Ad2AR-wt but not Ad2AR-Ile164 (Figure 2A). Importantly, ISO-mediated tubule formation was similar to that induced by VEGF, used as positive control.
Because angiogenesis is intimately associated to EC proliferation, we explored whether stimulation of 2ARs in ECs can increase cell number. In subconfluent bovine aorta ECs (BAECs), the addition of ISO caused a biphasic response with an initial increase, followed by a drop in cell number after 36 hours (Figure 2B). Loss of cell viability is probably related to multiple effects of ISO, which are dependent on time and dosage, because it can be observed after 24 hours, when using higher doses of ISO (>10eC8 mol/L; supplemental Figure I). The response to AR stimulation is largely dependent on endogenous 2AR because cell proliferation can be prevented by the selective 2AR inhibitor ICI 118 551 (ICI; Figure 2B). Consistent with an effect on cell proliferation, AR stimulation by ISO resulted in an increase in [3H]-thymidine incorporation (Figure 2C), an index of DNA synthesis, and phosphorylation of retinoblastoma (Rb) protein, which removes the inhibition of E2F in the nucleus causing the cell cycle progression from G1 to S23. This response in BAEC was attributable to 2AR signaling because it was attenuated by the 2AR antagonist ICI (Figure 2D).
For in vitro analysis of the effects of added wild-type or mutant (Ile164) 2AR density on EC function, adenoviral-mediated 2AR overexpression was obtained (Figure 2E), causing an increase in total AR density (from 29.7±0.9 to 55.5±1.4 fmol/mg and 55.2±1.4 fmol/mg protein in Ad2AR-wt and Ad2AR-Ile164, respectively). Overexpression of the wild-type but not the Ile164 mutant 2AR resulted in increased cell proliferation and DNA synthesis as measured by [3H]-thymidine incorporation, and ISO further enhanced these mitogenic responses (Figure 2B and 2C). The need of an intact 2AR signaling through the cAMP second messenger to achieve cell proliferation was verified by the antagonist Rp-cAMP because the presence of this inhibitor attenuated the proliferative response after 24 hours of ISO treatment (Figure 2F).
Angiogenesis is largely dependent on ERK/MAPK activation24eC26 because it can promote EC proliferation and expression of VEGF, which, in turn, sustains the proangiogenic phenotype.27 Using BAECs, we found that ISO leads to significant ERK activation (Figure 2G) and VEGF production (Figure 2H). Moreover, overexpression of the wild-type 2AR enhanced ERK/MAPK activation (Figure 2G). The 2AR-Ile164 failed to induce the potentiation of ERK/MAPK observed with the 2AR-wt (supplemental Figure II). To further assess the role of 2AR-mediated ERK/MAPK activation in BAEC proliferation, we used an inhibitor of ERK/MAPK activation, U0126 (10eC6 mol/L), and observed reduced BAEC proliferation in response to ISO (Figure 2F). Because 2ARs can stimulate VEGF production, we evaluated whether this cytokine is responsible for the proliferative effects of ECs after ISO. VEGF signaling was inhibited by using either an antibody to VEGF (200 ng/mL) or an inhibitor of the VEGF receptor. In both cases, BAEC proliferation in response to ISO was reduced (Figure 2F). These results suggest that 2AR-mediated EC proliferation is dependent, at least in part, on VEGF production and release.
Effects of 2AR Signaling on EC Apoptosis
Our cell proliferation data demonstrate that chronic exposure to ISO causes a loss in cell viability after 36 hours (Figure 2B). This is probably initiated by serum deprivation but is clearly further sustained by chronic AR activation. EC number may also be the result of cellular apoptosis under the control of ARs. Indeed, a previous report has shown that the 2AR in neonatal cardiac myocytes produces proapoptotic and antiapoptotic effects, and this signaling involves the p38 MAPK and PI3-kinase/Akt pathways.28 We have recently shown that in endothelium, 2ARs activate Akt.29 Other reports have documented the ability of ARs to stimulate p38 MAPK,28,30 which is considered an important mechanism of receptor-mediated apoptosis.31,32 Using BAECs, we first determined whether 2AR stimulation could result in Akt or p38 MAPK activation, and indeed, this was found with endogenous ARs as well as overexpressed 2ARs (Figure 3A and 3B). Next, we examined apoptosis using caspase-3 cleavage as a marker. Serum starvation and ISO reproducibly activated caspase-3 in BAECs, and both responses were significantly greater in ECs overexpressing the wild-type 2AR (Figure 3C). The involvement of p38 MAPK is suggested by the negative regulation of the p38/MAPK inhibitor SB203580 on 2AR-mediated caspase-3 cleavage (Figure 3D). Overall, these apoptosis data correlate with the observed reduction in cell number at 36 and 48 hours observed in cell proliferation experiments (Figure 2B).
Importantly, we also examined whether 2ARs have any effect on prosurvival pathways mediated by PI3-kinase/Akt. We repeated the above apoptosis assays in the presence of inhibitors of Akt activation and found that caspase-3 cleavage was significantly enhanced after ISO in Ad2AR-wt and control cells (Figure 3D; supplemental Figure III). Overall, our data show that 2ARs exert a dualistic effect on cell survival, with activation and inhibition of apoptosis through means of p38 MAPK and PI3-kinase/Akt, respectively.
Discussion
We verified that the 2AR is an endogenous mediator of angiogenesis in vivo, using a rat model of neovascularization and adrenergic activation attributable to hindlimb ischemia.14 The observed downregulation 2AR density suggests a role for this receptor in the compensatory response to chronic ischemia and adrenergic activation. Adenoviral-mediated overexpression of wild-type 2AR in vivo improves neoangiogenesis activity documented by digital imaging and perfusion techniques. Moreover, histological analysis showed that 2AR signaling preserves and enhances the number of capillaries in the ischemic area. The implications of this phenomenon were further tested in SHR, which experience impaired AR signaling and angiogenesis. In this model, the removal of the femoral artery causes severe perfusion deficit with necrosis and self-inflicted amputations of the ischemic limb. These alterations were all attenuated after intrafemoral artery delivery of the Ad2AR-wt. Mechanistic studies show that 2ARs control EC proliferation as well as stimulating proapoptotic and antiapoptotic pathways. These novel effects of 2ARs in ECs are in addition to their recently discovered role on NO release and regulation of vascular tone.5,29 AR stimulation activates proliferative and cell death and survival pathways, but the 2AR-mediated EC proliferative mechanism appears to be relevant for therapeutic angiogenesis.
It is noteworthy that 2ARs can induce cell proliferation and promote cell survival in other tissues; in particular, Kim et al recently described that 2ARs expressed on cardiac fibroblasts can induce cell proliferation through ERK/MAPK-dependent mechanisms.8 Our current data demonstrate that this signaling paradigm also exists in ECs because 2AR stimulation increased EC number, and this cellular proliferative effect is blocked by ERK/MAPK inhibition. Although we did not investigate the whole signal transduction involved in the proangiogenic phenotype induced by 2AR in ECs, our data are suggestive that cAMP production is important in this response.
2ARs exert a positive effect on EC ERK/MAPK activation by at least two mechanisms. First, stimulation of endothelial ARs and wild-type 2AR overexpression directly activate ERK/MAPK. Second, 2AR stimulation can induce the release of VEGF, which can also activate ERK/MAPK.26 ERK/MAPK inhibition totally prevents EC proliferation, demonstrating that this kinase is critical for AR-mediated cell mitogenesis and proliferation. The ability of ARs to induce VEGF production and release was reported previously for 1ARs and 3ARs in adipose tissue.33 Here, we show that this effect can also be demonstrated for the 2ARs found on ECs. Our in vitro results may appear in contrast with the in vivo observation of lower systemic VEGF in the Ad2AR-wt treated rats. On the contrary, both results testify the facilitation of angiogenesis mediated by an early boost of VEGF production that chronically causes a faster resolution of ischemia with the consequent return of systemic VEGF toward lower levels.
Concerning cell survival, Morisco et al have also shown in neonatal cardiomyocytes that 2AR signaling can prevent apoptosis through Akt activation.34 Zhu et al have confirmed this observation in adult cardiomyocytes.28 In ECs, too, 2AR activation leads to Akt-mediated apoptosis protection. Furthermore, in ECs, 2ARs can also stimulate apoptosis through a p38/MAPK mechanism. The net effect of proapoptotic and antiapoptotic signaling results in an eventual loss of cell number when ARs are chronically activated, whereas in the short term, there is an increase in cell number.
The effects of 2AR signaling on EC biology are clearly dependent on the overall integrity of the total signal transduction pathway. Indeed, overexpression of the naturally occurring Ile164 2AR mutant failed to induce the enhancement of neoangiogenesis observed in vivo in the ischemic hindlimb. This mutant receptor is largely nonfunctional and poorly activates downstream G-proteineCmediated signaling events. In transgenic mice with cardiac overexpression (&45-fold over total AR density) of this receptor, the changes in cardiac contractility induced by AR stimulation were not different from those of nontransgenic littermate.17 Furthermore, in patients harboring this polymorphism in heterozygosity, 2AR-dependent responses to exercise (cardiac index, stroke volume, systemic vascular resistance) were all reduced by 50%.19 In vitro, the EC50 of the mutant receptor to epinephrine is 5x higher than that of the wild type.16 Our results add to these data, showing that its overexpression did not alter the angiogenic response of ischemic hindlimb, nor in vitro EC proliferation, thymidine incorporation, and apoptosis. These results work also as a further control, ruling out the hypothesis of a "mass effect" because of overexpression-induced transactivation of non-2AR via heterodimerization.35 Our results imply that patients carrying this polymorphism may have an impairment of angiogenic response, in particular, in those conditions that are associated to sympathetic activation, such as chronic ischemia or cardiac dysfunction. Indeed, Liggett et al showed reduced survival in heart failure patients harboring the Ile164 mutant of the 2AR gene,36 thus suggesting a more profound inadequacy of the adaptive responses of these patients to the challenged hemodynamic condition. Our data provide the ground to interpret these findings as the result of the impaired angiogenesis associated to this polymorphism, leading to a reduced myocardial perfusion in a situation in which angiogenesis largely determines cardiac function and growth.37
The sympathetic nervous system regulates in vivo blood vessel growth, although this process was believed to be mediated by ARs and limited to vascular smooth muscle cell proliferation.38 Recently, another sympathetic neurotransmitter, the neuropeptide Y, has been demonstrated to be a potent angiogenic factor in vivo through means of the endothelial Y2 receptors.39 It is therefore emerging that the sympathetic nervous system is an important determinant of neoangiogenesis, acting through means of catecholamines and neuropeptides. As further support to this notion, mice lacking the ability to produce catecholamines are not able to fully develop during fetal life.40
In conclusion, we provide evidence that 2ARs are involved in the control of EC biology with implications in neoangiogenesis in response to ischemia. This is a novel finding carrying critical relevance in the current and future treatment of chronic ischemia. Therapeutic angiogenesis is the ultimate rescue of ischemic tissue, pursued using soluble growth factors such as VEGF, with the limitation of the potential facilitation of tumor genesis or growth. Our data suggest that therapeutic angiogenesis in ischemic tissue might be more efficiently and selectively achieved when associated to activation of 2AR signaling by means of available classical drugs or novel molecular tools such as adenoviral-mediated 2AR gene transfer.
Acknowledgments
This work was supported by grants from Italian Ministry of University and Research (B.T., G.I.), Fondazione Telethon Grant n. GGP04039 (L.P.) and National Institutes of Health grant R01 HL65360 (W.J.K.). The authors are indebted to Alberto Frezza and Giuseppe Rusciano for their valuable technical support.
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the Dipartimento di Medicina Clinica e Scienze Cardiovascolari ed Immunologiche (G.I., M.C., D.S., G.G., A.C., G.S., E.C., F.P., O.P., M.C., B.T.), Universite?Federico II, Napoli, Italy; Dipartimento di Biochimica e Biotecnologie Mediche (V.C., L.P., F.S.), Universite?Federico II, Napoli, Italy; CEINGE-Biotecnologie Avanzate (V.C., L.P., F.S.), Napoli, Italy; Dipartimento di Scienze Biomorfologiche e Funzionali (V.C., G.G.A.), Universite?Federico II, Napoli, Italy; Center for Translational Medicine (W.J.K.), Thomas Jefferson University, Philadelphia, Pa; and IRCCS Neuromed (B.T.), Pozzilli, Italy.
Abstract
2-Adrenergic receptors (2ARs) are widely expressed, although their physiological relevance in many tissues is not yet fully understood. In vascular endothelial cells, they regulate NO release and vessel tone. Here we provide novel evidence that 2ARs can regulate neoangiogenesis in response to chronic ischemia. We used in vivo adenoviral-mediated gene transfer of the human 2AR to the endothelium of the rat femoral artery and increased 2AR signaling resulting in ameliorated angiographic blood flow and hindlimb perfusion after chronic ischemia. Histological analysis confirmed that 2AR overexpression also produced benefits on capillary density. The same maneuver partially rescued impaired angiogenesis in spontaneously hypertensive rats (SHR), whereas gene delivery of the G-proteineCcoupling defective mutant Ile164 2AR failed to provide ameliorations. Stimulation of endogenous and overexpressed 2AR on endothelial cells in vitro was found to regulate cell number by inducing proliferation and [3H]-thymidine incorporation through means of extracellular receptor-activated kinase and vascular endothelial growth factor. The 2AR also has novel effects on endothelial cell number through stimulation of proapoptosis and antiapoptosis pathways involving p38 mitogen-activated protein kinase and PI3-kinase/Akt activation. Therefore, 2ARs play a critical role in endothelial cell proliferation and function including revascularization, suggesting a novel and physiologically relevant role in neoangiogenesis in response to ischemia.
Key Words: angiogenesis rats polymorphism hypertension in vivo digital angiography
Introduction
The endothelium controls several vascular functions, including vascular tone and permeability, thrombosis, hemostasis, and angiogenesis.1 It is noteworthy that all these functions can be regulated by activation of receptors, and often, the same receptor can activate multiple endothelial functions.
Adult angiogenesis only occurs in particular conditions such as wound healing, tumorogenesis, hypoxia, and chronic ischemia.2 It is a phenomenon intimately associated with endothelial cell (EC) proliferation, which appears to be under control of the extracellular receptor-activated kinase (ERK)/mitogen-activated protein kinase (MAPK)eCmediated signaling cascades.2 The most important system regulating angiogenesis is the cytokine vascular endothelial growth factor (VEGF), although a number of other cytokines and hormones acting through various tyrosine kinase and G-proteineCcoupled receptors are also implicated in this process.
The adrenergic system is the major regulator of cardiac and vascular function, and evidence is mounting for the relevance of this system in the control of endothelial vasodilation through means of 2- and -adrenergic receptors (ARs). In particular, 2ARs, the most abundant ARs in the vasculature,3,4 modulate the release of NO, causing endothelium-dependent vasodilation.5 2ARs are G-proteineCcoupled receptors activated by adrenergic catecholamines and promote a series of intracellular signal transduction pathways, leading to multiple cell-specific responses.6,7 Recently, it was proposed that 2ARs modulate cell proliferation, at least in fibroblasts, by activating ERK/MAPK through pathways dependent on -arrestins.8 The physiological implications of such an in vitro observation have not yet been fully investigated in vivo.
Altogether, the above considerations provide the background for the current investigation into the role of 2ARs in the control of angiogenesis. Accordingly, we evaluated whether 2ARs on ECs could enhance ischemia-induced angiogenesis in vivo. Also, in ECs, we evaluated the in vitro effects of 2AR overexpression on cell signaling and biology.
Materials and Methods
In Vitro and In Vivo Procedures
Experimental procedures were performed as described previously.5,9eC13 Extended details are provided in the online data supplement, available at http://circres.ahajournals.org.
Results
Effects of In Vivo 2AR Gene Transfer to the Endothelium During Chronic Ischemia in Wistar Kyoto Rats
We studied neoangiogenesis in a well-characterized rat model of chronic hindlimb ischemia in the rat.14 Our control group consisted of pooled data collected from sham-treated and AdEmpty viruseCtreated rats because no differences were found between these two treatments (supplemental Table I). In this model, 15 days after resection of the femoral artery, we observed a reduction in AR density within the ischemic hindlimb, which was attributable to a selective downregulation of 2ARs, the major AR subtype found in this tissue (Table). The numbers included in the Table reflect the sum of ARs expressed on all cell types present in the hindlimb area, including, but not solely, ECs. Also, 15 days after femoral artery resection, there was impairment in limb perfusion assessed by digital angiography (Figure 1A; supplemental video 1). These hemodynamic changes were paralleled by the rarefaction in capillary (<5 e蘭) density in the anterior tibial muscle compared with the contralateral normal limb (Figure 1C and 1D). In a subset of rats, we injected the Ad2AR-wt into the femoral artery at the time of surgery. Consistent with previous results,15 this resulted in 2AR overexpression in muscle, arterial capillaries, and venous (Figure 1E), which was double that of endogenous AR density found in the rat hindlimb (Table). After 15 days of ischemia, rats receiving Ad2AR-wt had significantly improved hindlimb perfusion with increased blood flow compared with untreated rats (Figure 1A and 1B; supplemental video 2). Similarly, histological analysis of the anterior tibial muscle revealed restored capillary density because of Ad2AR-wt treatment (Figure 1C and 1D). These positive benefits in vivo appear to be attributable to enhanced 2AR signaling because delivery of Ad2AR-Ile164 (Figure 1A through 1D) resulted in no beneficial effects on hindlimb hemodynamic or histology. The Ile1642AR mutation is a naturally occurring human polymorphism that causes a severe impairment in the G-protein coupling.16eC19
Chronic ischemia leads to increased systemic VEGF levels, a potent proangiogenesis cytokine, that typically return to baseline values once the ischemia is eliminated.20,21 We examined VEGF levels in our model in the blood and in the nonischemic contralateral hindlimb. In both samples, VEGF-165 levels were significantly lower in ischemic rats treated with Ad2AR-wt compared with control and Ad2AR-Ile164 rats, suggesting that 2AR overexpression significantly lessens ischemia (Figure 1F and 1G). Overall, these results suggest that 2ARs can regulate key endothelial cellular functions in vivo in response to chronic ischemia.
Impaired Angiogenesis Ameliorated by In Vivo 2AR Gene Transfer to the Endothelium During Chronic Ischemia in Spontaneously Hypertensive Rats
To further explore the role of endothelial 2AR in pathophysiological conditions, we repeated the in vivo experiment using the angiogenesis-impaired spontaneously hypertensive rat (SHR).22 Previously in this model, we have shown that reduced vascular and endothelial AR signaling and vasodilation is corrected by 2AR gene transfer.5 Fifteen days after resection of the femoral artery in SHR, there was a dramatic impairment of hindlimb perfusion, evidenced by the elevated occurrence of blistering, necrosis, or self-inflicted amputation of the ischemic paw (occurrence of necrosis, amputation, and blistering: SHR 35%; Wistar Kyoto [WKY] 0%; P<0.05; 2 test). This loss of perfusion was confirmed by digital angiography (Figure 1A), dyed microsphere dilution (Figure 1B), and by histological analysis (Figure 1D). In SHR treated with the AD2AR-wt, we observed a partial correction of this phenotype indicated by the reduced occurrence of blistering (10%; P<0.05; 2 test) and the ameliorated hemodynamic and histological parameters (Figure 1A, 1B, and 1D).
Finally, the analysis by Western blot of VEGF performed on the contralateral nonischemic hindlimb showed that systemic levels of this cytokine were reduced in the Ad2AR-wt SHR compared with the nontreated group, suggesting that local ischemia was resolved (Figure 1G).
Effects of 2AR Signaling on EC Proliferation
Our in vivo results do not let us discern whether the protective effects of 2AR overexpression in the rat hindlimb arterial endothelium after ischemic damage are attributable to an effect on cell proliferation or cell survival. These mechanistic questions were addressed in vitro using ECs in culture. The overall angiogenic properties of 2AR were recapitulated under conditions in which stimulated human ECs organize in network forming tubules. The AR agonist isoproterenol (ISO) increased the number of connections among endothelial tubules in vitro, and this response was magnified by Ad2AR-wt but not Ad2AR-Ile164 (Figure 2A). Importantly, ISO-mediated tubule formation was similar to that induced by VEGF, used as positive control.
Because angiogenesis is intimately associated to EC proliferation, we explored whether stimulation of 2ARs in ECs can increase cell number. In subconfluent bovine aorta ECs (BAECs), the addition of ISO caused a biphasic response with an initial increase, followed by a drop in cell number after 36 hours (Figure 2B). Loss of cell viability is probably related to multiple effects of ISO, which are dependent on time and dosage, because it can be observed after 24 hours, when using higher doses of ISO (>10eC8 mol/L; supplemental Figure I). The response to AR stimulation is largely dependent on endogenous 2AR because cell proliferation can be prevented by the selective 2AR inhibitor ICI 118 551 (ICI; Figure 2B). Consistent with an effect on cell proliferation, AR stimulation by ISO resulted in an increase in [3H]-thymidine incorporation (Figure 2C), an index of DNA synthesis, and phosphorylation of retinoblastoma (Rb) protein, which removes the inhibition of E2F in the nucleus causing the cell cycle progression from G1 to S23. This response in BAEC was attributable to 2AR signaling because it was attenuated by the 2AR antagonist ICI (Figure 2D).
For in vitro analysis of the effects of added wild-type or mutant (Ile164) 2AR density on EC function, adenoviral-mediated 2AR overexpression was obtained (Figure 2E), causing an increase in total AR density (from 29.7±0.9 to 55.5±1.4 fmol/mg and 55.2±1.4 fmol/mg protein in Ad2AR-wt and Ad2AR-Ile164, respectively). Overexpression of the wild-type but not the Ile164 mutant 2AR resulted in increased cell proliferation and DNA synthesis as measured by [3H]-thymidine incorporation, and ISO further enhanced these mitogenic responses (Figure 2B and 2C). The need of an intact 2AR signaling through the cAMP second messenger to achieve cell proliferation was verified by the antagonist Rp-cAMP because the presence of this inhibitor attenuated the proliferative response after 24 hours of ISO treatment (Figure 2F).
Angiogenesis is largely dependent on ERK/MAPK activation24eC26 because it can promote EC proliferation and expression of VEGF, which, in turn, sustains the proangiogenic phenotype.27 Using BAECs, we found that ISO leads to significant ERK activation (Figure 2G) and VEGF production (Figure 2H). Moreover, overexpression of the wild-type 2AR enhanced ERK/MAPK activation (Figure 2G). The 2AR-Ile164 failed to induce the potentiation of ERK/MAPK observed with the 2AR-wt (supplemental Figure II). To further assess the role of 2AR-mediated ERK/MAPK activation in BAEC proliferation, we used an inhibitor of ERK/MAPK activation, U0126 (10eC6 mol/L), and observed reduced BAEC proliferation in response to ISO (Figure 2F). Because 2ARs can stimulate VEGF production, we evaluated whether this cytokine is responsible for the proliferative effects of ECs after ISO. VEGF signaling was inhibited by using either an antibody to VEGF (200 ng/mL) or an inhibitor of the VEGF receptor. In both cases, BAEC proliferation in response to ISO was reduced (Figure 2F). These results suggest that 2AR-mediated EC proliferation is dependent, at least in part, on VEGF production and release.
Effects of 2AR Signaling on EC Apoptosis
Our cell proliferation data demonstrate that chronic exposure to ISO causes a loss in cell viability after 36 hours (Figure 2B). This is probably initiated by serum deprivation but is clearly further sustained by chronic AR activation. EC number may also be the result of cellular apoptosis under the control of ARs. Indeed, a previous report has shown that the 2AR in neonatal cardiac myocytes produces proapoptotic and antiapoptotic effects, and this signaling involves the p38 MAPK and PI3-kinase/Akt pathways.28 We have recently shown that in endothelium, 2ARs activate Akt.29 Other reports have documented the ability of ARs to stimulate p38 MAPK,28,30 which is considered an important mechanism of receptor-mediated apoptosis.31,32 Using BAECs, we first determined whether 2AR stimulation could result in Akt or p38 MAPK activation, and indeed, this was found with endogenous ARs as well as overexpressed 2ARs (Figure 3A and 3B). Next, we examined apoptosis using caspase-3 cleavage as a marker. Serum starvation and ISO reproducibly activated caspase-3 in BAECs, and both responses were significantly greater in ECs overexpressing the wild-type 2AR (Figure 3C). The involvement of p38 MAPK is suggested by the negative regulation of the p38/MAPK inhibitor SB203580 on 2AR-mediated caspase-3 cleavage (Figure 3D). Overall, these apoptosis data correlate with the observed reduction in cell number at 36 and 48 hours observed in cell proliferation experiments (Figure 2B).
Importantly, we also examined whether 2ARs have any effect on prosurvival pathways mediated by PI3-kinase/Akt. We repeated the above apoptosis assays in the presence of inhibitors of Akt activation and found that caspase-3 cleavage was significantly enhanced after ISO in Ad2AR-wt and control cells (Figure 3D; supplemental Figure III). Overall, our data show that 2ARs exert a dualistic effect on cell survival, with activation and inhibition of apoptosis through means of p38 MAPK and PI3-kinase/Akt, respectively.
Discussion
We verified that the 2AR is an endogenous mediator of angiogenesis in vivo, using a rat model of neovascularization and adrenergic activation attributable to hindlimb ischemia.14 The observed downregulation 2AR density suggests a role for this receptor in the compensatory response to chronic ischemia and adrenergic activation. Adenoviral-mediated overexpression of wild-type 2AR in vivo improves neoangiogenesis activity documented by digital imaging and perfusion techniques. Moreover, histological analysis showed that 2AR signaling preserves and enhances the number of capillaries in the ischemic area. The implications of this phenomenon were further tested in SHR, which experience impaired AR signaling and angiogenesis. In this model, the removal of the femoral artery causes severe perfusion deficit with necrosis and self-inflicted amputations of the ischemic limb. These alterations were all attenuated after intrafemoral artery delivery of the Ad2AR-wt. Mechanistic studies show that 2ARs control EC proliferation as well as stimulating proapoptotic and antiapoptotic pathways. These novel effects of 2ARs in ECs are in addition to their recently discovered role on NO release and regulation of vascular tone.5,29 AR stimulation activates proliferative and cell death and survival pathways, but the 2AR-mediated EC proliferative mechanism appears to be relevant for therapeutic angiogenesis.
It is noteworthy that 2ARs can induce cell proliferation and promote cell survival in other tissues; in particular, Kim et al recently described that 2ARs expressed on cardiac fibroblasts can induce cell proliferation through ERK/MAPK-dependent mechanisms.8 Our current data demonstrate that this signaling paradigm also exists in ECs because 2AR stimulation increased EC number, and this cellular proliferative effect is blocked by ERK/MAPK inhibition. Although we did not investigate the whole signal transduction involved in the proangiogenic phenotype induced by 2AR in ECs, our data are suggestive that cAMP production is important in this response.
2ARs exert a positive effect on EC ERK/MAPK activation by at least two mechanisms. First, stimulation of endothelial ARs and wild-type 2AR overexpression directly activate ERK/MAPK. Second, 2AR stimulation can induce the release of VEGF, which can also activate ERK/MAPK.26 ERK/MAPK inhibition totally prevents EC proliferation, demonstrating that this kinase is critical for AR-mediated cell mitogenesis and proliferation. The ability of ARs to induce VEGF production and release was reported previously for 1ARs and 3ARs in adipose tissue.33 Here, we show that this effect can also be demonstrated for the 2ARs found on ECs. Our in vitro results may appear in contrast with the in vivo observation of lower systemic VEGF in the Ad2AR-wt treated rats. On the contrary, both results testify the facilitation of angiogenesis mediated by an early boost of VEGF production that chronically causes a faster resolution of ischemia with the consequent return of systemic VEGF toward lower levels.
Concerning cell survival, Morisco et al have also shown in neonatal cardiomyocytes that 2AR signaling can prevent apoptosis through Akt activation.34 Zhu et al have confirmed this observation in adult cardiomyocytes.28 In ECs, too, 2AR activation leads to Akt-mediated apoptosis protection. Furthermore, in ECs, 2ARs can also stimulate apoptosis through a p38/MAPK mechanism. The net effect of proapoptotic and antiapoptotic signaling results in an eventual loss of cell number when ARs are chronically activated, whereas in the short term, there is an increase in cell number.
The effects of 2AR signaling on EC biology are clearly dependent on the overall integrity of the total signal transduction pathway. Indeed, overexpression of the naturally occurring Ile164 2AR mutant failed to induce the enhancement of neoangiogenesis observed in vivo in the ischemic hindlimb. This mutant receptor is largely nonfunctional and poorly activates downstream G-proteineCmediated signaling events. In transgenic mice with cardiac overexpression (&45-fold over total AR density) of this receptor, the changes in cardiac contractility induced by AR stimulation were not different from those of nontransgenic littermate.17 Furthermore, in patients harboring this polymorphism in heterozygosity, 2AR-dependent responses to exercise (cardiac index, stroke volume, systemic vascular resistance) were all reduced by 50%.19 In vitro, the EC50 of the mutant receptor to epinephrine is 5x higher than that of the wild type.16 Our results add to these data, showing that its overexpression did not alter the angiogenic response of ischemic hindlimb, nor in vitro EC proliferation, thymidine incorporation, and apoptosis. These results work also as a further control, ruling out the hypothesis of a "mass effect" because of overexpression-induced transactivation of non-2AR via heterodimerization.35 Our results imply that patients carrying this polymorphism may have an impairment of angiogenic response, in particular, in those conditions that are associated to sympathetic activation, such as chronic ischemia or cardiac dysfunction. Indeed, Liggett et al showed reduced survival in heart failure patients harboring the Ile164 mutant of the 2AR gene,36 thus suggesting a more profound inadequacy of the adaptive responses of these patients to the challenged hemodynamic condition. Our data provide the ground to interpret these findings as the result of the impaired angiogenesis associated to this polymorphism, leading to a reduced myocardial perfusion in a situation in which angiogenesis largely determines cardiac function and growth.37
The sympathetic nervous system regulates in vivo blood vessel growth, although this process was believed to be mediated by ARs and limited to vascular smooth muscle cell proliferation.38 Recently, another sympathetic neurotransmitter, the neuropeptide Y, has been demonstrated to be a potent angiogenic factor in vivo through means of the endothelial Y2 receptors.39 It is therefore emerging that the sympathetic nervous system is an important determinant of neoangiogenesis, acting through means of catecholamines and neuropeptides. As further support to this notion, mice lacking the ability to produce catecholamines are not able to fully develop during fetal life.40
In conclusion, we provide evidence that 2ARs are involved in the control of EC biology with implications in neoangiogenesis in response to ischemia. This is a novel finding carrying critical relevance in the current and future treatment of chronic ischemia. Therapeutic angiogenesis is the ultimate rescue of ischemic tissue, pursued using soluble growth factors such as VEGF, with the limitation of the potential facilitation of tumor genesis or growth. Our data suggest that therapeutic angiogenesis in ischemic tissue might be more efficiently and selectively achieved when associated to activation of 2AR signaling by means of available classical drugs or novel molecular tools such as adenoviral-mediated 2AR gene transfer.
Acknowledgments
This work was supported by grants from Italian Ministry of University and Research (B.T., G.I.), Fondazione Telethon Grant n. GGP04039 (L.P.) and National Institutes of Health grant R01 HL65360 (W.J.K.). The authors are indebted to Alberto Frezza and Giuseppe Rusciano for their valuable technical support.
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