Desert Hedgehog Promotes Ischemia-Induced Angiogenesis by Ensuring Peripheral Nerve SurvivalNovelty and Significance
Rationale: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves.
Objective: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia.
Methods and Results: We induced hindlimb ischemia in wild-type and Dhh–/– mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh–/–. By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle.
Conclusions: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.
Blood vessels and nerves have a similar organization pattern in the human body. Recent investigations revealed that they share many common mechanisms and signals for growth, patterning, and survival.1–4 Moreover, studies performed in mice suggest that angiogenesis is, at least in part, regulated by guidance signals derived from nerves. In the skin, blood vessel branching and arterial differentiation are determined by Schwann cell–derived signals.5 Similarly, in the mouse retina, astrocyte-derived vascular endothelial growth factor A (VEGFA) has been shown to guide endothelial tip cells.6 In the skeletal muscle, nerves were shown to be necessary to maintain blood vessels in adults.7 Conversely, vascular cells and, more particularly, smooth muscle cells have been shown to produce Artemin, a neurotrophic factor for sympathetic nerve axons.8 In the brain, activated endothelial cells (ECs) produce brain-derived neurotrophic factor, which promotes migration and recruitment of neurons.9
Evidence suggesting modulation of angiogenesis and vasculogenesis by Hedgehog (Hh) signaling emerged in studies of vascularization of embryonic tissues. Because each of the 3 Hh proteins has a unique expression pattern and because their expression does not overlap,10 Sonic hedgehog (Shh) and Indian hedgehog were shown to regulate angiogenesis in specific tissues during embryogenesis.11,12 On the contrary, Desert hedgehog (Dhh) never has been involved in angiogenesis.
Dhh is expressed by Schwann cells of peripheral nerves and controls structural and functional integrity of adult nerves.13 As a consequence, Dhh deficiency in mice and in humans has been associated with peripheral neuropathy.14,15 In this tissue, Dhh was shown to regulate EC function by controlling vessel permeability.14 In adults, previous investigations indicated that Hh signaling is necessary to maintain coronary vasculature in physiological conditions.16 Moreover, inhibition of Hh protein activity by the neutralizing 5E1 antibody administration impairs ischemia-induced angiogenesis.16,17 Because 5E1 antibody blocks the activity of the 3 Hh ligands,18 the specific role of each Hh protein in the regulation of blood vessel homeostasis has not been established. We hypothesized that Dhh, which is specifically expressed by peripheral nerves, is critical for ischemia-induced angiogenesis, and we performed a series of experiments to further characterize the role of Dhh during angiogenesis and ischemic tissue repair. We found that Dhh is necessary for angiogenesis; however, it does not modulate EC function. On the contrary, it promotes peripheral nerve survival in the setting of ischemia. In parallel, we demonstrated that peripheral nerves express proangiogenic factors and are necessary for ischemia-induced angiogenesis demonstrating that Dhh regulates angiogenesis by promoting peripheral nerve survival in the setting of ischemia.
C57BL/6 mice were obtained from Charles River Laboratories and bred in our animal facility. Dhh heterozygote mice19 under CD1-C57BL/6 genetic background were provided by M. Wijgerde (Erasmus University Medical Center, Erasmus University of Rotterdam, Rotterdam, the Netherlands). Dhh+/– mice were bred together to obtain Dhh–/– mice and wild-type (WT) control mice. Pups were genotyped as described previously.20 SmoFlox,21 Rosa26R, and Ptch1-LacZ mice22 were obtained from the Jackson Laboratory. Pdgfb-CreERT2 mice23 were kindly provided by M. Fruttiger and bred with SmoFlox mice. Gli1-CreERT2 mice24 were kindly provided by A.L. Joyner (Developmental Biology Program, Center for Stem Cell Biology, Sloan-Kettering Institute, New York, NY) and bred with Rosa26R mice. Mice were handled in accordance with the guidelines established by the National Institute of Medical Research (Institut National de la Santé et de la Recherche Médicale) and approved by the local institutional animal care and use committee. Animals were anesthetized by 2.5% to 4.0% isoflurane inhalation or by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Mice were administrated 1 mg/kg buprenorphine 30 minutes before surgical procedures. Cre recombinase was activated by intraperitoneal injection of 1 mg tamoxifen (Sigma) for 5 consecutives days.
Hindlimb Ischemia Model and Assessments
Hindlimb ischemia (HLI) was performed as described25 previously in 8- to 12-week-old mice. Briefly, the left femoral artery and all of the side branches were dissected and excised from the proximal end of the femoral artery to the distal portion of the saphenous vein.
For histological assessment and gene expression analysis, mice were euthanized and tibialis anterior muscles were harvested and then cut in half; the lower half was fixed in methanol, paraffin-embedded, and cut into 6-µm sections; and the upper half was snap-frozen in liquid nitrogen. Each group included 3 to 15 animals. Capillary density was evaluated in sections stained for the expression of CD31.
For microcomputed tomography vasculature imaging, mice were perfused with a mixture containing 80% neoprene latex (Neoprene Latex Dispersion 671 A; DuPont, France) and barium sulfate powdered to 1 μm (3 g/mL, MicrOpaque oral solution [Guerbet, France]) as described previously.26 The vasculature was imaged using a high-resolution microcomputed tomography imaging system (eXplore Locus SP; General Electric Healthcare). Image acquisitions and reconstructions were performed using Scan Control and Reconstruction Utility programs. Data were acquired in an axial mode covering a single hindlimb, as described previously.26 The quantification parameters were obtained using the Microview ABA program.
For X-gal staining, mice were perfused with LacZ fix solution (phosphate-buffered saline containing 0.2% glutaraldehyde, 5 mmol/L EGTA, and 100 mmol/L MgCl2) before tibialis anterior muscles were harvested. Cryosections (10-µm-thick) were prepared and postfixed for 10 minutes in 0.2% glutaraldehyde and then stained overnight at 37°C in X-gal staining solution (1 mg/mL X-gal; Sigma). Tissue sections were counterstained with nuclear fast red solution (Sigma).
The cDNA encoding the N terminal part of Dhh (ie, the signal sequence plus the entire sequence of the processed form of Dhh) was amplified from MS1 cell cDNA with the following primers: 5′-AGGACAAGAACGCTCCCTTC-3′ and 5′ GAGCGGATCCATTTCCCGGAAATCAGCCTC-3′. The N terminal part of Dhh cDNA was subsequently digested with NcoI and BamHI and cloned into pIRES2-EGFP plasmid (Clontech) digested with SmaI and BamHI.
Gene therapy was performed in 12-week-old mice. Four days after HLI surgery was performed, mice were randomly assigned to receive 200 µg pIRES-EGFP or 200 µg pIRES-NDhh together with 0.05% pluronic, as described previously.27 The DNA/pluronic mix was injected intramuscularly in the tibialis anterior muscle. Mice were euthanized 10 days after HLI surgery was performed.
The dorsal skin of the thigh was cut and the posterior muscles were divided to show the sciatic nerve. Denervation of the left leg was obtained by cutting and removing 5 mm of the sciatic nerve.
ECs were identified with rat anti-CD31 antibodies (BD Pharmingen). Dhh was stained with goat anti-C-terminal Dhh (R&D Systems) antibodies. Schwann cells were identified with rabbit anti-S100 antibodies (Dako) or rat anti-myelin basic protein antibodies (Abcam). Neurotrophin (NTF) 3, VEGFA, and angiopoietin (Angpt) 1 were stained using rabbit anti-NTF3 antibodies (Abbiotec), rabbit anti-VEGFA antibodies (Abcam), and goat anti-Angpt1 antibodies (Santa Cruz), respectively. For immunofluorescence analyses, primary antibodies were resolved with Alexa-Fluor–conjugated secondary antibodies (Invitrogen), and nuclei were counterstained with 4',6-diamidino-2-phenylindole (1/5000). For immunohistochemical analyses, primary antibodies were sequentially stained with biotin-conjugated secondary antibodies (Vector) and streptavidin-horseradish peroxidase complex (Amersham), and then the stain was developed with a 3,3'-diaminobenzidine substrate kit (Vector Laboratories). Tissues were counterstained with hematoxylin.
Quantitative Reverse-Transcription Polymerase Chain Reaction
RNAs were isolated by using Tri Reagent (Molecular Research Center) as instructed by the manufacturer from 3×105 cells or from skeletal muscle that had been snap-frozen in liquid nitrogen and homogenized. For quantitative reverse-transcription polymerase chain reaction analyses, total RNA was reverse-transcribed with Moloney murine leukemia virus reverse-transcriptase (Promega), and amplification was performed on a DNA Engine Opticon 2 (MJ Research) using B-R SYBER Green SuperMix (Quanta Biosciences). Primer sequences are reported in Online Table I.
Absolute quantification of cDNA copy number was achieved using plasmid DNA standards (ie, Hh encoding plasmids described above or pGEM-T plasmids (Promega) in which we cloned the corresponding polymerase chain reaction amplicon). The relative expression of each mRNA was calculated by the comparative threshold cycle method and normalized to hypoxanthine guanine phosphoribosyl-transferase mRNA expression.
Isolation of Nerve Cells
Mice were euthanized by cervical dislocation and their sciatic nerves were harvested. Adjacent connective tissues and perineurium were stripped off under a dissecting microscope. Nerves were digested in an enzymatic solution containing 0.2% collagenase NB4 (Worthington) for 1 hour at 37°C. An equal volume of 0.05% trypsin–EDTA was then added, and nerve segments were mechanically disaggregated by vigorous pipetting for 3 to 5 minutes. After centrifugation, Schwann cells were plated in poly-l-lysine (Sigma) plus laminin-coated (Sigma) dishes and cultured in DMEM/F12 culture medium containing 10% fetal bovine serum, 2 µmol/L Forskolin (Sigma), and 10 ng/mL Heregulin-β1 (Promocell).
To isolate perineurial fibroblasts, nerves were harvested, digested, and cultured following the same protocol except that perineurium was not stripped off and cells were passaged at least once before being used to get rid of most Schwann cells.
Cell Viability Assay Using Propidium Iodide
A total of 40 000 cells were seeded in each well of a 24-well plate. The day after, cell death was induced by a 24-hour incubation with 100 µmol/L to 1 mmol/L H2O2. To identify dead cells, culture medium was removed and replaced by phosphate-buffered saline containing 2 µg/mL of propidium iodide. Cells were incubated with propidium iodide for ≥5 minutes on ice. Propidium iodide–positive cells were identified by fluorescent microscopy.
Results are reported as mean±standard error of the mean (SEM). Comparisons between groups were analyzed for significance with the nonparametric Mann-Whitney test. Differences between groups were considered to be significant when P≤0.05.
Dhh Is the Main Hh Expressed During the Angiogenic Phase of Ischemic Muscle Repair
To investigate whether Dhh expression is detected and modulated during skeletal muscle ischemia-induced angiogenesis, HLI was surgically induced in mice. Tibialis anterior muscle tissue was harvested before and 2, 5, 7, 10, 14, and 21 days after HLI surgery. Consistent with previous investigations,16,17,28 we found that Shh is strongly upregulated (by 80-fold vs control) 2 days after HLI surgery was performed, but its expression returned rapidly to baseline from day 5 after surgery. Interestingly, Dhh mRNA expression, which is ≈10-times higher than that of Shh and Indian hedgehog in healthy skeletal muscle, was quite stable until day 7, increased by ≈2-fold from day 10 after HLI (P=0.036), and remained high until muscle repair was completed (Figure 1A). Indian hedgehog expression remains very low during the entire process of ischemic muscle repair (Figure 1A). In conclusion, Dhh, with its role in angiogenesis never investigated before, is the most expressed Hh during the angiogenesis phase of ischemic muscle repair (ie, from day 5 to day 10).
According to previous investigations,10,20,29 we found Dhh to be expressed by S100-positive peripheral nerves in the healthy muscle and in the regenerating ischemic muscle (2, 5, and 10 days after HLI surgery was performed; Figure 1B). Thus, we hypothesized that Schwann cell–derived Dhh regulates ischemia-induced angiogenesis.
Dhh Promotes Ischemia-Induced Angiogenesis
The role of Dhh in ischemia-induced angiogenesis was assessed by using the mouse HLI model.25 HLI was surgically induced in Dhh–/– mice and in their WT littermates. The leg recovery was evaluated 10 days after surgery was performed. The mean clinical score of necrosis of the ischemic foot of Dhh–/– mice was 2.18±0.26, whereas it was 0 for all of the WT mice (Figure 2A and 2B). Increased foot necrosis in Dhh–/– mice was correlated with significantly decreased tissue perfusion (Figure 2C and 2D) and delayed muscle repair (Figure 2E and 2F and Online Figure I), demonstrating an important role for Dhh in skeletal muscle repair.
Revascularization of the ischemic leg was first evaluated by microcomputed tomography imaging (Figure 3A–3D). Artery density was dramatically lower in Dhh–/– mice when compared with WT mice (the number of arteries per micrometer cubed in Dhh–/– was 0.026±0.005 vs 0.111±0.010; P=0.042 in WT mice; Figure 3B). The mean diameter of vessels also was significantly lower in Dhh–/– mice (33±1 µm vs 50±3 µm in WT mice; P=0.042; Figure 3C). Moreover, organization of the vessel network was strongly impaired in Dhh–/– mice, because vessel connectivity was only 53±11 in Dhh–/–, whereas it was 227±19 in WT mice (P=0.042; Figure 3D). Those results were confirmed by CD31 staining of tibialis anterior muscle sections (Figure 3E). The number of CD31+ vessels per millimeter squared was 66±6 in Dhh–/– mice vs 164±11 in WT mice (P=0.002; Figure 3F). Altogether these data demonstrate for the first time that Dhh is critical for ischemia-induced angiogenesis in the setting of skeletal muscle tissue repair.
To confirm the specific role of Dhh in angiogenesis in adults, Dhh expression was rescued via gene therapy in Dhh knockout mice. Unilateral HLI was surgically induced in WT and Dhh–/– mice. Plasmids encoding either green fluorescent protein alone (pIRES-EGFP) or Dhh together with green fluorescent protein (pIRES-NDhh) were administered in the tibialis anterior muscle 4 days after surgery. This time point was chosen because it comes immediately before the angiogenesis phase of ischemic muscle repair. Dhh overexpression was detected from 5 to 10 days after surgery (data not shown). Dhh gene therapy significantly enhances ischemia-induced angiogenesis in Dhh knockout mice because capillary density in the muscle of Dhh–/– mice treated by Dhh was comparable with that of WT mice (162±9 CD31+ vessels/mm2 in Dhh–/– mice vs 172±7 in WT mice). These later data then confirmed the role of Dhh during muscle repair.
ECs Do Not Mediate Hh-Dependent Angiogenesis in the Setting of Ischemia
With the aim to investigate the direct role of Dhh on ECs in the setting of ischemic muscle repair, unilateral HLI was induced in Pdgfb-CreERT2-SmoFlox/Flox mice and in their SmoFlox/Flox control littermates. Smoothened (Smo) recombination was induced by tamoxifen injections 1 week before HLI surgery was performed. The activity of the Pdgfb promoter–driven Cre recombinase in ECs of both healthy and ischemic skeletal muscles was verified using Rosa26R mice (Online Figure II). Revascularization of the ischemic leg was evaluated 5 and 10 days after HLI surgery was performed. Neither feet perfusion measured after laser Doppler perfusion imaging (Figure 4A–4C) nor capillary density in the tibialis anterior muscle (Figure 4D and 4E) was different in Pdgfb-CreERT2-SmoFlox/Flox or in their control SmoFlox/Flox littermates. Consistently, skeletal muscle repair was not delayed in Pdgfb-CreERT2-SmoFlox/Flox mice, and the clinical score of necrosis of their ischemic foot was equal to 0. Dhh does not promote ischemia-induced angiogenesis by regulating EC function directly.
Dhh Does Not Modulate Proangiogenic Factor Expression in Nerve Fibroblasts
To identify cells in which Hh signaling is active in the ischemic skeletal muscle, we used Gli1-CreERT2-Rosa26R and Ptch1-LacZ reporter mice. Hh proteins are known to interact with their specific receptor Patched-1, which decreases the transmembrane protein Smo and activates Gli transcription factors. Gli activation induces expression of downstream target genes, including Patched-1 and Gli1.30 As shown in Online Figure IIIA, Hh canonical pathway activity (ie, Gli1 and Patched-1 expression) is almost exclusively detected in perineurial cells, which indicates that Dhh expressed by Schwann cells mainly regulates perineurial fibroblast physiology.
Thus, we verify whether Dhh regulates proangiogenic factor expression in perineurial fibroblasts. As shown in Online Figure IIIB through IIIE, although Dhh significantly activates Gli1 mRNA expression in nerve-derived fibroblasts, it does not modulate VEGFA, Angpt1, or NTF3 mRNA expression. Those results suggest that, in contrast to Shh,31 Dhh does not promote angiogenesis by upregulating VEGFA, Angpt1, or NTF3.
Peripheral Nerve–Derived Signals Are Necessary to Drive Ischemia-Induced Angiogenesis
Because Dhh was shown to be necessary in peripheral nerve integrity and regeneration in adults,13,14 we hypothesized that Dhh signaling to perineurial fibroblasts promotes nerve survival in the setting of ischemia and, by doing, so maintains the pool of proangiogenic factors expressed by nerves. To test this hypothesis, we first verified whether peripheral nerves within the regenerating skeletal muscle express proangiogenic factors. To this aim, we performed immunostaining of sections of ischemic skeletal muscles harvested 5 days after HLI was induced. We found that nerves within the skeletal muscle produce VEGFA, Angpt1, and NTF3 (Online Figure IV).
We then verified whether nerve-derived signals are necessary to drive ischemia-induced angiogenesis. To this aim, mice were denervated by removing 5 mm of the left sciatic nerve immediately after HLI was performed. Mice were euthanized 10 days after HLI surgery. First, we verified that nerve density was significantly reduced in the regenerating muscle of denervated mice compared with undenervated mice (Online Figure V). Consistent with our hypothesis, CD31 staining of capillaries demonstrated that angiogenesis is significantly reduced in denervated mice compared with the sham-operated (undenervated) control (capillary density was 87±6 vessels/mm2 in denervated mice vs 153±14 in sham-operated mice; Figure 5A and 5B). Moreover, delayed angiogenesis was correlated with a significant decrease of VEGFA, Angpt1, and NTF3 (Figure 5C and 5D). Similar to what we found in Dhh–/– mice, impaired angiogenesis observed in denervated mice induced a significant reduction of feet perfusion (Figure 6A and 6B) and delayed skeletal muscle regeneration (Figure 6C and 6D). As a consequence, the clinical score of necrosis was 2.44±0.18 in denervated mice, whereas it was mainly 0 in undenervated mice (Figure 6E and 6F).
Dhh Promotes Peripheral Nerve Survival in the Setting of Ischemia
We then tested whether nerve survival was impaired in the absence of Dhh. We measured S100+ peripheral nerve density in the tibialis anterior muscle of Dhh–/– mice and of WT mice 0, 2, 5, and 10 days after HLI was performed. As shown in Figures 6B and 7A, in physiological conditions (ie, before HLI surgery was performed), nerve density was not different in Dhh–/– and WT mice. On the contrary, 2 days after surgery, whereas the number of nerves/mm2 only slightly decreased (from 6.44±2.03 to 5.64±0.17 nerves/mm2) in WT mice, it significantly decreased in Dhh–/– mice (from 6.41±1.99 to 3.18±0.18/mm2), showing that Dhh promotes peripheral nerve survival after an ischemic injury. To confirm this result, we cocultured Schwann cells and fibroblasts from the sciatic nerves of Dhh–/– and WT mice (Online Figure VIA). Schwann cells were used here as the source (or not) of Dhh (Online Figure VIB) and, as shown in Online Figure VIC, Schwann cell–derived Dhh stimulated Hh signaling pathway in fibroblasts. Among Dhh–/– cells, the number of propidium iodide–positive dying fibroblasts was significantly higher than among WT cells when cell death was induced by 0.5 or 0.8 mmol/L of H2O2 (Figure 7C and 7D). This result demonstrates that Dhh promotes nerve fibroblast survival.
The present article demonstrates for the first time to our knowledge the crucial role of Dhh in the setting of skeletal muscle ischemia-induced angiogenesis. Moreover, this study shows that Dhh promotes angiogenesis by ensuring peripheral nerve survival and, indirectly, growth factor production, thus identifying an original regulation of ischemia-induced angiogenesis.
The embryonic Hh signaling pathway was shown to be reactivated in adult injured tissues ≈10 years ago.31 More particularly, Shh expression has been shown to be strongly increased by skeletal muscle injury, including surgically induced ischemia, mechanical crush, and cardiotoxin injection.16,17,28 This pathway was shown to be necessary for ischemia-induced angiogenesis by using the Hh ligand–blocking antibody 5E1.17 We show, for the first time to our knowledge, that, except for the early neutrophil infiltration step of muscle repair in which Shh is transiently expressed, the main Hh ligand expressed in the limb skeletal muscle is Dhh. Moreover, Dhh expression is increased during the delayed phase of muscle repair corresponding with the regeneration of the vascular network, suggesting a possible role for this protein in vessel growth, which is consistent with the antiangiogenic effect of 5E1 anti-Hh antibody observed in the mouse model of HLI.17
In this study, we used Dhh-deficient mice to specifically investigate the role of Dhh. Our data reveal, for the first time to our knowledge, that Dhh has a major role in ischemia-induced angiogenesis and muscle repair. This effect of Dhh is different from that of Shh, because Shh overexpression observed 2 days after HLI surgery in WT mice (Figure 1) occurs at the same level in Dhh-deficient mice (data not shown).
Hh signaling was proposed to promote angiogenesis via, at least in part, upregulation of several proangiogenic factors, including VEGFA, Angpt1, and stromal cell-derived factor 1 in mesenchymal fibroblasts or in myocytes.31–33 We found that, in contrast to Shh, Dhh does not stimulate VEGFA, Angpt1, or NTF3 expression in fibroblasts. Consistent with what has been observed in embryos34 and, what has been suggested in adults,31 we demonstrated that Dhh does not promote angiogenesis via a direct activation of ECs. On the contrary, the present study reveals that Dhh promotes peripheral nerve survival in the ischemic muscle, demonstrating a new mechanism of action of Hh proteins to promote angiogenesis and muscle repair.
Mechanisms underlying tissue regeneration that have been widely studied in amphibians, such as urodeles, have revealed the essential role of peripheral nerves in this process.35 More particularly, Schwann cells were shown to produce secreted signals called AG (for anterior gradient necessary for limb regeneration). In mammals, denervation has been shown to impair ear lobe regeneration after injury induced by an ear punch.35 Accordingly, we found that denervation of the leg, immediately after ischemia was induced, severely impairs ischemia-induced angiogenesis and muscle regeneration.
Interestingly, peripheral nerves were shown to produce proangiogenic factors in the developing skin, and signals from Schwann cells have been shown to determine blood vessel network organization.5 Neurotrophins were shown to promote ischemia-induced angiogenesis.36,37 As a consequence, we and others found that proangiogenic factor expression, including Angpt1 and VEGFA, is diminished in denervated muscles.38,39
In conclusion, the present study, supported by data from others, demonstrates the essential role of the peripheral nerve as a source of proangiogenic factors in the setting of ischemia-induced angiogenesis. As a consequence, factors regulating peripheral nerve physiology and ensuring their survival, such as Dhh, might be interesting targets to consider for therapeutic angiogenesis, at least in the setting of ischemia.
Finally, such a concept is supported by observations made in patients with neuropathy. One study reported that severity of neuropathy was the most important factor associated with the development and recurrence of foot ulcers in diabetic patients,40 and another reported that the somatic and autonomic nerve alterations may play a relevant role in the progression of the disease toward critical limb ischemia.41
The authors thank Jérôme Guignard (Institut National de la Santé et de la Recherche Médicale U1034, Pessac) for his excellent technical assistance in the animal facility. The authors thank A. L. Joyner for providing Gli1-CreERT2 mice. CreERT2 mice are licensed by Groupement d'Intérêt Economique-Centre Européen de Recherche en Biologie et en Médecine (Institut Clinique de la Souris-Institut de Génétique et de Biologie Moléculaire et Cellulaire).
Sources of Funding
This study was supported by grants from the Fondation de la Recherche Médicale program on cardiovascular aging (DCV20070409258); the Conseil Régional d’Aquitaine (action inter-régionale Aquitaine-Midi Pyrénées); the Communauté de Travail des Pyrénées and the Agence Nationale de la Recherche program (ANR-07-PHYSIO-010-02 to A.-P. Gadeua, E. Traiffort); and the National League Against Cancer (to M.-A. Renault and M. Ruat). C. Chapouly and S. Vandierdonck are supported by fellowships from the Centre Hospitalier Universitaire de Bordeaux.
In December 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.300871/-/DC1.
- desert hedgehog
- endothelial cell
- hindlimb ischemia
- sonic hedgehog
- vascular endothelial growth factor A
- Received July 29, 2011.
- Revision received January 21, 2013.
- Accepted January 23, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Embryonic signaling pathways participate in ischemic tissue repair in adults.
Shh promotes angiogenesis by upregulating proangiogenic factors including vascular endothelial growth factor A (VEGFA).
Peripheral nerves drive tissue regeneration in amphibians and regulate angiogenesis and blood vessel network architecture during development.
What New Information Does This Article Contribute?
Peripheral nerves contribute to ischemia-induced angiogenesis.
Dhh, a Schwann cell–derived protein, is essential for structural and functional integrity of peripheral nerves. It is also crucial for ischemia-induced angiogenesis by promoting peripheral nerve survival in ischemic conditions.
This study shows that nerve survival maintains proangiogenic nerve-derived factor levels and thereby promotes muscle repair. Our findings suggest that factors regulating peripheral nerve physiology and survival, such as Dhh, are important regulators of ischemia-induced angiogenesis and might be important targets to consider for therapeutic angiogenesis.