Targeted Delivery of Carbaprostacyclin to Ischemic Hindlimbs Enhances Adaptive Remodeling of the Microvascular NetworkNovelty and Significance
Prostacyclin and its stable analogs play an important vascular protective role by promoting angiogenesis, but their role in arteriolar growth is unclear. Here, we examined the effect of prostacyclin stable analog carbaprostacyclin on arteriolar growth in mouse hindlimb ischemia. Using an osmotic-controlled release system to continuously deliver carbaprostacyclin or saline (control) to ischemic mouse hindlimbs for up to 14 days, we found that blood perfusion was significantly better at 7 and 14 days in carbaprostacyclin-treated mice than in saline-treated mice. Microscopic examination of the microvasculature showed more morphological signs of arteriolar formation in carbaprostacyclin- versus saline-treated legs. A double-blind, quantitative microcomputed tomography analysis indicated that carbaprostacyclin-treated legs had markedly increased vascular volume and small- to medium-sized vessel numbers that correspond to decreased vessel separation. A proteome profiler antibody array demonstrated that carbaprostacyclin-treated ischemic muscles secreted significantly higher amounts of acidic fibroblast growth factor and other chemokines. Conditioned media containing those secreted factors promoted smooth muscle cell growth and migration. Additionally, increased acidic fibroblast growth factor protein levels were detected in smooth muscle cells and skeletal myotubes at different time periods after carbaprostacyclin treatment. Furthermore, the selective peroxisome proliferation-activated receptor β/δ antagonist significantly suppressed carbaprostacyclin-induced acidic fibroblast growth factor protein production. Collectively, our data provide the first morphological and molecular evidence that local delivery of carbaprostacyclin promotes vascular growth in hindlimb ischemia, and that peroxisome proliferation-activated receptor β/δ signaling plays a critical role in inducing acidic fibroblast growth factor expression.
- acidic fibroblast growth factor
- hindlimb ischemia
- peroxisome proliferator-activated receptor β/δ
Critical limb ischemia (CLI) occurs in response to arterial occlusion, which leads to insufficient blood supply to the lower extremity.1,2 Conventional treatments for CLI are less effective when CLI progresses and causes obstruction of arterioles. In such cases, patients may develop untreatable claudication, rest pain, and ulcers that can progress to gangrene and other infections requiring amputation of the lower limb.3,4 New therapeutic approaches are needed to promote arteriolar growth, which potentially could reroute nutrient blood to the ischemic region and reduce peripheral resistance distal to the occlusion.
Prostacyclin (PGI2) has several favorable properties that could be used in CLI therapy. As a vasoactive drug, PGI2 is an important mediator of vascular homeostasis5,6 and also inhibits thrombosis and platelet aggregation. Because PGI2 is chemically unstable and has a short circulating half-life of 1 to 2 minutes, stable analogs have been developed for clinical use.7,8 PGI2 and its stable analogs exert their actions by interacting with the surface PGI2 receptor (IP) or nuclear peroxisome proliferator-activated receptor β/δ (PPARβ/δ).9–11 Although PGI2 limits inward vessel remodeling, such as neointima formation and atherosclerosis, little is known about its action on adaptive outward remodeling such as growth of arterioles. Furthermore, we and others have shown the positive effect of PGI2 and its stable analogs on angiogenesis,12,13 in which the PPARβ/δ signaling pathway plays a critical role. However, the specific role of PGI2 in adaptive arteriolar growth is unknown.
In the current study, we created a hindlimb ischemia model to study the vascular protective effect of the stable PGI2 analog and PPARβ/δ agonist carbaprostacyclin (cPGI2). We tested the hypothesis that cPGI2 promotes arteriolar growth in hindlimb ischemia. To achieve the maximal drug effect within the target tissue, we used an osmotically controlled release system to selectively deliver cPGI2 or saline (control) to the ischemic leg. We used multiple imaging techniques to assess the vascular effects of cPGI2 compared with saline on the microvascular network of ischemic legs. Our results demonstrated that the constant local release of cPGI2 in ischemic hindlimbs promotes vascular remodeling. In addition, we showed that the cPGI2-PPAR β/δ axis is involved in promoting arteriolar growth.
Detailed methods are available in the online-only Data Supplement.
cPGI2 Treatment Improves Perfusion and Promotes Arteriolar Growth in Hindlimb Ischemia
To examine whether cPGI2 enhances perfusion, we compared the efficacy of local delivery of cPGI2 or saline in restoring blood flow in ischemic hindlimbs. We measured blood perfusion before and after ligation of the femoral artery, 24 hours after treatment, and up to 14 days thereafter. Figure S1 in the online-only Data Supplement shows the representative color-coded images and quantitative raw data analysis of hindlimb perfusion. At 7 days after surgery, laser Doppler-derived data showed that blood perfusion was significantly better in cPGI2-treated mice than in saline-treated mice (58.80±5.74% versus 40.60±3.14%, respectively; P<0.05; n=5/group; Figure 1A); this finding also held true at 14 days (88.40±8.71% [cPGI2 group] versus 54.60±6.67% [saline group]; P<0.01; n=5/group; Figure 1A). There were no significant differences (P>0.05) between groups in systolic pressure, mean arterial pressure, heart rate, or tail blood volume before or during the treatment period (Figure S2).
In live mice, we microscopically examined the dynamic changes in the microvascular morphology of the limb region distal to the ligation where cPGI2 was applied. Three days after treatment, more microvascular rearrangement was observed in the cPGI2-treated group than in the saline-treated group (Figure S3). Consistent with our 3-day findings, we noted more distinct structural remodeling at the arteriolar level in the cPGI2 group than in the saline group at 7 days (Figure S4), including tortuosity of arteriolar-to-arteriolar connections (white arrows, Figure S4D versus S4B, respectively), and increased intersecting of adjacent arterioles (blue arrows, Figure S4D versus S4B) in cPGI2-treated legs. Arteriolar networks were identified by their branching out from a large feeder femoral artery and from the saphenous branch of the descending genicular artery.
Because PGI2 and its analogs are short-acting vasodilators,14 we used 2 independent approaches to evaluate whether the different configuration of vascular structures between cPGI2- and saline-treated ischemic legs was a result of cPGI2-induced vasodilation. First, we simultaneously applied cPGI2 to the anterolateral thigh muscle of the left limb and saline to the right limb in the same mouse for 24 hours; ischemia was not induced in either hindlimb. Interestingly, microscopic examination of live mice at 24 hours showed no visible differences in the preexisting microvascular network of cPGI2-treated legs compared with saline-treated contralateral legs (Figure S5A and S5B). Moreover, we did not observe a corkscrew-like pattern of arterioles or well-developed arteriolar connections. In the second approach, we compared the vasculature in the same ischemic anterolateral thigh before and 20 minutes after the constant application of cPGI2. As seen in the first approach, the acute application of cPGI2 did not enhance the visibility of the vasculature, and the morphology was similar with and without cPGI2 administration (Figure S5C and S5D). Together, our data suggest that arteriolar growth, not vasodilation, accounts for the differences observed in the vascular network in cPGI2-treated ischemic hindlimbs at 3 and 7 days.
cPGI2 Treatment Positively Affects Remodeling of the Microvascular Network in Ischemic Hindlimbs
We used high-definition, volumetric, quantitative micro-computed tomography (CT) to assess the overall microvascular geometry of ischemic and contralateral nonischemic legs at 14 days after femoral occlusion and constant local administration of cPGI2 or saline in ischemic legs. Bones were decalcified during sample preparation to eliminate interference during visualization of the microvasculature. We evaluated 4 different morphological properties in a double-blind manner. Supporting the perfusion data presented above, we found that the vascular volume of cPGI2-treated legs was significantly higher than that of saline-treated legs (41.28±2.22 versus 27.11±2.85 mm3; P<0.05; n=5/group; Figure 1B). In addition, cPGI2-treated legs had significantly more blood vessels (0.16±0.014 versus 0.09±0.011 1/mm; P<0.05; n=5/group; Figure 1C) and less vessel separation (distance between vessels, 6.60±0.52 versus 10.15±1.14 mm; P<0.05; n=5/group; Figure 1D) than did saline-treated legs. These findings suggest better development of the vascular system in the cPGI2-treated group than in the saline-treated group. We also found a positive but not significant effect on average connectivity (n=5/group; Figure 1E) in cPGI2 versus saline groups. Similar global morphometric analyses were used to evaluate contralateral nonischemic legs. No significant differences were observed between cPGI2 and saline groups in any of the 4 morphological variables (Figure S6A–S6D).
To further verify the proarteriogenic effect of cPGI2, we generated a quantitative histogram by using micro-CT to illustrate the frequency and distribution of blood vessel size in cPGI2-treated and saline-treated ischemic legs and contralateral nonischemic legs. Compared with saline-treated ischemic legs, cPGI2-treated ischemic legs showed a significant increase in small vessels, with vessel diameter bins ranging from 40 to 60 μm (P<0.05; n=5/group; Figure 1F). These data strongly indicate that cPGI2 improves perfusion, in part, by arteriogenic rather than dilatory effects because dilation would cause an increase in the diameter of all-sized vessels, not just small vessels.15 Representative micro-CT images of vessel remodeling of cPGI2- and saline-treated limbs showed that vascular remodeling is more prominent in the region of cPGI2 delivery than in the similar anatomic location of saline delivery (Figure 1G). We similarly evaluated contralateral nonischemic legs and found no significant differences in vessel distribution in the cPGI2 and saline groups (Figure S6E). Figure S6F shows representative micro-CT images of the vasculature in contralateral nonischemic limbs of cPGI2- and saline-treated groups. Together, these data indicate that increased vessel formation is an important means by which cPGI2 improves perfusion in ischemic legs.
Acidic Fibroblast Growth Factor and Other Cytokines Released From cPGI2-Treated Ischemic Hindlimbs Increase Smooth Muscle Cell Growth and Migration
To help determine how cPGI2 affects arteriolar growth, we performed a proteome profiler array to assess the simultaneous secretion of chemokines and soluble factors from ischemic thigh tissues 3 days after treatment with cPGI2 or saline (Figure S7). Of the 53 factors measured in the array (Figure 2A), acidic fibroblast growth factor (fibroblast growth factor-1, FGF1), insulin-like growth factor binding protein-1, pentraxin-3, and plasminogen activator inhibitor-1 were significantly increased in cPGI2-treated thigh muscles as compared with saline-treated samples (n=3/group; Figure 2B). Collectively, our array results suggest that multiple mediators within the local environment may affect vessel growth.
Because vascular smooth muscle cells (SMCs) are the major cellular component of arterioles, we evaluated the paracrine effect of factors released by cPGI2- and saline-treated ischemic thigh tissue on the proliferation and migration of SMCs. SMCs were cultured for 48 hours in conditioned medium (CM) from cPGI2- or saline-treated ischemic thigh tissues; the number of SMCs was significantly higher after treatment with cPGI2-CM than with saline-CM (Figure 2C). We then used a scratch wound migration assay to determine the effects of CM on SMC migration. Incubation with cPGI2-CM significantly increased SMC migration over that seen with saline-CM (Figure 2D and 2E). Thus, cPGI2 treatment created a favorable microenvironment for SMC growth and migration.
cPGI2 Induces Expression of FGF1 in Skeletal Myotubes and Vascular SMCs Under Hypoxia
Because our array showed an increase in FGF1 levels in cPGI2-treated limb tissues and FGF1 seems to promote the growth of arterioles in hindlimb ischemia,16 we focused our investigation on how cPGI2 regulates FGF1 and on the types of cells that are responsible for increased FGF1 levels. Myofibers are the main component of limb muscles; therefore, we used C2C12 myotubes to study cPGI2-mediated FGF1 expression. To mimic in vivo ischemia, we conducted these experiments in a hypoxic environment (1.5% O2). At 2 hours after cPGI2 treatment, FGF1 protein levels were similar in cPGI2-treated and untreated cells. However, the expression of FGF1 in untreated cells was transient, peaking at 4 hours, and a rapid decrease was detected at 8 hours. In contrast, FGF1 expression was sustained in cPGI2-treated cells, persisting for up to 8 hours (Figure 3A). We also evaluated the effects of cPGI2 on FGF1 expression in vascular SMCs under similar hypoxic conditions. Treatment of SMCs with cPGI2 resulted in a more acute effect than that seen in myotubes; a marked increase in FGF1 protein levels was observed 2 hours after the addition of cPGI2 as compared with untreated SMCs (Figure 3B). An FGF1-positive lysate was used in parallel during Western blot (Figure S8).
Because dual cellular pathways are involved in cPGI2-induced biological activities, we examined whether cell surface prostacyclin receptor (IP) signaling or nuclear receptor PPARβ/δ signaling is responsible for the increased FGF1 expression. Specific receptor antagonists were used to selectively target cPGI2-induced IP or PPARβ/δ signaling in C2C12 myotubes and SMCs. Although the IP receptor antagonist CAY-10441 (1 µmol/L) attenuated FGF1 expression in C2C12 myotubes (P>0.05) 8 hours after cPGI2 (10 µmol/L) treatment, the PPARβ/δ antagonist GSK3787 (1 µmol/L) exerted a more significant suppression on cPGI2-induced FGF1 upregulation (P<0.05; Figure 3C). In SMCs, the IP receptor antagonist CAY-10441 did not affect FGF1 protein levels induced by cPGI2 after 2-hour treatment. However, similar treatment with the PPARβ/δ antagonist GSK3787 significantly decreased FGF1 expression (Figure 3D). Together, our data suggest a novel role of cPGI2-PPARβ/δ signaling in regulating FGF1 protein levels in both skeletal myotubes and SMCs under hypoxia.
cPGI2 Treatment Increases Nascent Vessel Formation in Ischemic Hindlimbs
To convert nascent vascular tubes into functional vasculature, supporting mural cells must be recruited to encircle the newly formed endothelial tube and ensure vessel survival. Mural cells interact with luminal endothelial cells of newly formed microvessels to preserve the integrity of the abluminal barrier and to facilitate cell–cell communication for vessel stabilization and maturation. To understand the role of cPGI2 on mural cell recruitment during vessel growth, we performed immunofluorescence staining to localize endothelial and mural cells within the microvasculature. We used anti–von Willebrand factor antibody to label the abluminal endothelial surface and anti-neuron-glial antigen 2 (NG2) antibody to label mural cells. NG2 is a transmembrane proteoglycan expressed exclusively by mural cells during development of the neovasculature.17 We used laser scanning confocal microscopy to examine NG2+ mural cells around the luminal endothelial layer 7 days after arterial ligation. We found more NG2+ vessels in cross sections of anterior thigh muscles in cPGI2-treated mice than in saline-treated mice (Figure 4A and 4B). Interestingly, most of the NG2+ arterioles were located in subcutaneous tissue and were seen underneath the muscle; these are the areas where cPGI2 was delivered (sustaining high cPGI2 concentrations) and where active vascular remodeling occurred. Quantitative analysis indicated that the number of NG2+ microvessels ranging in size from 15 to 50 µm in diameter was significantly higher in the cPGI2 group than in the saline group (38.00±2.41/high-power field versus 18.69±2.12/high-power field; P<0.01; n=3/group; Figure 4C). To further confirm that the NG2+ cells were mural cells, we performed α-smooth muscle actin (SMA) staining and found that NG2+ vessels >15 µm in diameter coexpressed SMA (Figure S9). Together, our data suggest that the controlled release of cPGI2 in ischemic hindlimbs increased the formation of NG2+ vessels.
In the current study, we provide evidence that local cPGI2 delivery facilitates arteriolar growth in hindlimb ischemia. We compared perfusion changes in ischemic legs after vascular occlusion and constant cPGI2 or saline delivery and found that the relative perfusion was significantly better at 7 and 14 days in cPGI2-treated legs than in saline-treated legs. Furthermore, we showed that cPGI2 treatment resulted in a more elaborate microvascular structure at the arteriolar level, distal to the ligation site, than did saline treatment. Improved blood flow in the cPGI2 group coincided with the emergence of arteriolar anastomoses, including the development of arteriolar loops adjoining arterioles and collateral vessels connecting parallel neighboring arterioles. The structural remodeling response to cPGI2 was further confirmed by double-blind micro-CT analyses. Finally, we indicated a positive role of cPGI2-PPAR β/δ signaling in regulating FGF1 protein levels in vascular SMCs and C2C12 myotubes under hypoxia.
Because cPGI2 is a vessel dilator,18 we evaluated its dilatory effects on microvascular morphology. The hemodynamic response to prostacyclin has been shown to be measurable after a 24-hour continuous infusion of PGI214; therefore, we believe that our time point of measuring after 24 hours of cPGI2 exposure should be sufficient to view the vasodilatory effects of cPGI2 but would allow only minimal vascular growth. Interestingly, our microscopic observations showed that cPGI2 did not induce visible vasodilation; we found a similar network of microvascular structures in both cPGI2- and saline-treated legs. This finding indicates that cPGI2-induced dilatory effects cannot be seen by en face observation. Moreover, to minimize the effect of inherited morphological microvascular discrepancies between mice, we compared the microvascular morphology of left and right legs from the same mouse after local delivery of either cPGI2 or saline. Again, the application of cPGI2 in the ischemic anterolateral thigh for 20 minutes did not induce a change in the vasculature or lead to enhanced microvascular emergence when compared with the same vascular area before cPGI2 application. To our knowledge, no published studies have shown that cPGI2 can produce profound vessel relaxation resulting in a morphological change detectable via light microscopy. The vessel dilatory effect of cPGI2 has been previously observed by measuring the arteriolar response at 80 mm Hg intravascular pressure ex vivo in a vessel chamber administered with cPGI2.19 In support of the arteriogenic role of cPGI2 shown by live animal imaging, quantitative micro-CT demonstrated that cPGI2 delivery resulted in a significant increase in the number of vessels with diameters ranging from 40 to 60 µm. This finding is the first to demonstrate cPGI2-induced arteriolar remodeling that resulted in improved perfusion in ischemic legs.
In the current report, we limited our study of cPGI2 to an acute ischemia model. An animal model that mimics the effects of the chronic ischemic disease that occurs in patients would enable us to better elucidate the therapeutic effects of cPGI2. However, creating a mouse model with chronic ischemic limbs is challenging. In the presence of arterial occlusion, angiogenesis and arteriogenesis occurs to compensate for perfusion loss.20 Although we created severe ischemia after double ligation of the common femoral artery proximal to the origin of the femoral bifurcation, the average perfusion without drug treatment reached 54.6% at day 14 after surgery (saline-treated group, Figures 1A and S1). This compensatory blood recovery prevents us from examining drug function in a chronic limb ischemia model. To provide a prolonged window of time to investigate the therapeutic effects of cPGI2, we treated mice with cPGI2 immediately after surgery when minimal perfusion is observed. Using mice with a diminished ability of vascularization may enable us to establish a stable chronic ischemia model for testing drug effects.
To better understand how cPGI2 promotes microvascular remodeling in ischemic limbs, we performed a proteome profiler antibody array to screen for the expression of arteriogenic factors. Our results indicated that cPGI2 upregulates the local release of FGF1 from ischemic muscles at 3 days after femoral artery ligation. We further validated the array results by examining the effects of cPGI2 exposure on FGF1 protein levels in C2C12 myotubes and SMCs under hypoxia. We detected the presence of higher amounts of FGF1 protein in both cell types at different times after cPGI2 treatment. Overexpression of FGF1 has been shown to promote the growth of arterioles in mice by increasing the number and density of branches.21 In a hamster model of peripheral arterial disease, FGF1 gene transfer promoted arteriogenesis in ischemic hindlimbs.16 However, FGF1 has low thermal stability in the absence of heparin, is easily degraded by proteases, and has a short half-life in vivo.22 Thus, the instability of FGF1 limits its biological action in therapeutic applications. Our data suggest that administration of cPGI2 may help maintain a constant FGF1 concentration for an extended period, which may compensate for FGF1’s instability and increase its efficacy during the critical window of vascular remodeling.
We examined the signaling pathway involved in cPGI2-induced FGF1 protein expression under hypoxia. Our results indicate a novel role for PPARβ/δ signaling in promoting arteriolar growth. PPARβ/δ is a ligand-activated transcriptional factor that modulates target gene expression. Evidence has emerged suggesting a potential role for PPARβ/δ in prostacyclin-induced vascular protection.13 Although the proangiogenic property of stable PGI2 analogs is attributed to PPARβ/δ activity,23 little is known about how the PGI2-PPARβ/δ axis affects the growth or remodeling of preexisting arteriolar networks. cPGI2 is ideal for studying this issue because it functions as a stable PGI2 analog and is known to be an effective PPARβ/δ agonist. Here, we first used multiple quantitative imaging techniques to illustrate the positive effects of cPGI2 on arteriolar growth. We then found that a combination of soluble factors produced in the local tissue microenvironment of cPGI2-treated legs promotes SMC migration and growth. Furthermore, using a selective and irreversible PPARβ/δ inhibitor GSK3787, we demonstrated the positive effects of cPGI2-PPARβ/δ axis on the production of arteriogenic factor FGF1. Our results indicate a role for PPARβ/δ in adaptive vascular remodeling and suggest that PPARβ/δ may be a new therapeutic target for treating CLI.
Different mechanisms may be involved in cPGI2-induced FGF1 expression in different cell types. In SMCs, increased amounts of FGF1 were detected at 2 hours after cPGI2 treatment. This acute response depended on PPARβ/δ signaling because GSK3787 blocked the increase; this finding indicates the possibility that PPARβ/δ directly regulates the FGF1 gene. A recent study showed that the FGF1 gene promoter region contains a conserved PPAR response element.24 Thus, PPARβ/δ might act through transcriptional activation of FGF1. Interestingly, in C2C12 myotubes, a prolonged period of time (8 hours) was required for cPGI2 to exert a positive effect on FGF1 protein expression. Moreover, treatment of myotubes with the PPARβ/δ antagonist GSK3787 did not completely block the increase in FGF1 levels. Considering that similar treatment with the IP antagonist CAY 10441 also decreased cPGI2-induced FGF1 protein levels (although statistically not significant), it is possible that IP signaling is, in part, responsible for increased FGF1 production. Therefore, a more complex mechanism involving interaction between IP and PPARβ/δ signaling may affect the upregulation of FGF1 in skeletal myotubes. In the current study, we demonstrated a novel association between cPGI2-PPARβ/δ-FGF1 signaling and arteriolar growth in ischemic hindlimbs. However, we did not rule out additional mechanisms (eg, nitric oxide pathway) that may affect cPGI2-induced arteriolar growth.
Given the finding that the secreted arteriogenic factor FGF1 is persistently present in skeletal myotubes treated with cPGI2, the paracrine action of skeletal myofibers in promoting the formation of vasculature is noteworthy. Cytokines and growth factors released from inflammatory and vascular cells stimulate vascular growth, but the role of myofibers is poorly understood. Because myofibers are a major cell type surrounding arterioles in skeletal muscles, their signaling to neighboring vascular cells (eg, endothelial cells and SMCs) may affect arteriolar growth. Such signaling may not only reestablish a sufficient vascular network to provide fuel for myofibers but also reduce myofiber necrosis during ischemia. We postulated that the paracrine action of skeletal muscles triggered by therapeutic agents in ischemic conditions is an efficient strategy to potentiate arteriolar growth and to rapidly compensate for insufficient blood supply by recruiting preexisting arteriolar networks. Supporting this idea, our findings indicate that myotubes serve as a critical effector of cPGI2 signaling and as a hub for secreting the arteriogenic factor FGF1.
In conclusion, we present comprehensive evidence that cPGI2 positively affects adaptive vascular remodeling in hindlimb ischemia. We also provide evidence that cPGI2 promotes FGF1 protein expression in SMCs and skeletal myotubes via PPARβ/δ signaling. Because PGI2 is known to inhibit vascular dysfunction (eg, thrombosis and atherosclerosis), we believe our findings will be useful in developing novel treatment strategies for atherosclerotic diseases such as CLI.
CLI is the severe obstruction of blood flow to the lower extremities and is caused by adverse cardiovascular conditions such as atherosclerosis, hypertension, and hypercholesterolemia. Revascularization is the best treatment strategy for CLI. Prostacyclin and stable analogs are clinically recommended agents for treating CLI and have shown beneficial effects. In the current study, we used a local drug delivery system and demonstrated a novel function of the stable prostacyclin analog carbaprostacyclin (cPGI2) in promoting revascularization in hindlimb ischemia. Using multiple imaging techniques, we showed that cPGI2 attenuates hindlimb ischemia by promoting arteriolar growth. Moreover, we uncover a novel mechanism for cPGI2-enhanced vascular remodeling. We demonstrate cPGI2-induced nuclear receptor PPARβ/δ signaling positively affects FGF1 protein levels in vascular SMCs and skeletal myotubes. Our data suggest that cPGI2 may improve the paracrine action of ischemic skeletal myofibers to enhance arteriolar growth. Because ischemia results in blood loss at the microvascular level and arterioles directly control blood distribution throughout the hindlimbs, the cPGI2-PPAR β/δ axis may serve as an effective target for inducing arteriolar growth and increasing blood flow to ischemic tissue beds. Together, the current findings will advance our understanding of the prostacyclin-mediated PPARβ/δ signaling that controls adaptive vascular remodeling in hindlimb ischemia.
We thank Keith Michel of the MD Anderson Small Animal Imaging Facility for his technical assistance with micro-computed tomography imaging, Dr Edward T.H. Yeh for surgical assistance, Dr Darren Woodside for critical comments on this project, and Dr Rebecca A. Bartow for editorial assistance.
Sources of Funding
This work was supported, in part, by the American Heart Association National Scientist Development Grant.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.00458/-/DC1.
- Received October 19, 2012.
- Revision received February 21, 2013.
- Accepted February 22, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is New?
cPGI2 administered directly to the ischemic tissue bed improved perfusion by inducing arteriolar growth and remodeling and promoted the release of acidic fibroblast growth factor.
Nuclear receptor peroxisome proliferator-activated receptor β/δ signaling may be the primary pathway involved in arteriolar remodeling.
What Is Relevant?
Because of our aging population, critical limb ischemia is a major therapeutic challenge. Prostacyclin and its stable analogs are a therapeutic choice for treating critical limb ischemia, but the role of prostacyclin analogs in arteriolar growth is unknown.
The current study facilitates further understanding of the cPGI2-peroxisome proliferator-activated receptor β/δ axis in promoting arteriolar growth and helps to formulate a new strategy for the development of therapeutic agents.
Prostacyclin and stable analogs have shown beneficial effects in treating patients with critical limb ischemia. By using multiple imaging techniques, we have shown here that a stable prostacyclin analog, carbaprostacyclin (cPGI2), improves local perfusion by enhancing the growth of arteriolar networks. Our results provide the first evidence that the cPGI2-peroxisome proliferator-activated receptor β/δ axis positively affects acidic fibroblast growth factor protein expression in skeletal myotubes and smooth muscle cells. Together, the data suggest local delivery of cPGI2 exerts its therapeutic effects at the level of the arteriolar circulation.