Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia.
The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP–/–, global natriuretic peptide receptor (NPR)-B–/– and NPR-C–/– animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease.
Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP–/– and NPR-C–/–, but not NPR-B–/–, mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of G i, ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level.
These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.
The morbidity and mortality associated with peripheral artery disease (PAD) is an expanding unmet medical need as a consequence of the ageing population and growing prevalence of metabolic disorders. PAD affects >200 million individuals worldwide and typically manifests as intermittent claudication, but often progresses to, or even presents as, critical limb ischemia (CLI) characterized by pain, ulceration, and gangrene; many patients face amputation and early death. The pathology is triggered by atherosclerotic vascular occlusions and is underpinned by an insufficient angiogenic (hypoxia-triggered de novo blood vessel formation) and arteriogenic (shear stress–induced remodeling of the collateral network) response, compensatory and complementary mechanisms invoked to reinstate blood supply to the ischemic tissue. A central pathway coordinating both processes is driven by vascular endothelial growth factor (VEGF)-A, which preclinical studies indicated was a potential treatment for PAD. However, large-scale clinical trials using VEGF-A to promote angiogenesis and arteriogenesis in patients with PAD and CLI have proven disappointing. Therefore, delineation of the mechanisms and signaling pathways that underlie these intrinsic, restorative pathways is critical if the therapeutic potential of a proangiogenic strategy is to be harnessed.
The endothelium is pivotal in triggering angiogenesis, vascular remodeling, and minimizing functional deficit after ischemia. Endothelium-derived C-type natriuretic peptide (CNP) plays a fundamental role in regulating vascular homeostasis; CNP controls local blood flow in the resistance vasculature and systemic blood pressure, reduces the reactivity of leukocytes and platelets, and prevents the development of atherogenesis and aneurysm. Several facets of CNP biology suggest it may play a central role in angiogenesis. For instance, 2 of the primary stimuli for CNP release from endothelial cells are shear stress and transforming growth factor β, both of which are well-validated triggers for angiogenesis and arteriogenesis. In addition, hypoxia-inducible factor 1α, a fundamental driver of the ischemic angiogenic response, is a potent enhancer of natriuretic peptide expression, in particular, in cardiomyocytes. Our previous studies revealed that pulmonary microvascular endothelial cells from endothelium-specific CNP–/– (ecCNP–/–) and global NPR-C–/– mice proliferate significantly more slowly than wild-type (WT) cells, implying that CNP has a physiological role in regulating growth. The peptide also slows neointimal hyperplasia and promotes reendothelialization in vein grafts, in damaged carotid arteries, and after balloon angioplasty. CNP also maintains capillary density after myocardial infarction and hindlimb ischemia. Although these observations intimate that pharmacological administration of CNP may promote arteriogenesis and angiogenesis, previous work has reported that CNP blocks VEGF signaling to attenuate angiogenesis in vitro, and reduces sponge implant neovascularization in vivo, demonstrating that the function of CNP in this setting remains unclear and requires further investigation. Moreover, there is no evidence demonstrating that endothelium-derived CNP either directly stimulates angiogenesis or triggers endothelial cell processes critical for angiogenesis. Therefore, given recent findings showing an important role for CNP in vascular homeostasis, we sought to investigate whether endogenous CNP is a key regulator of angiogenesis and vascular remodeling.
All murine studies conformed to the UK Animals (Scientific Procedures) Act of 1986 and had approval from the local Animal Welfare and Ethical Review Body within Bart’s and The London School of Medicine. The human tissue studies were permitted under Local Research Ethics Committee decision 16/WA/0198 (Integrated Research Application System project ID: 193340) with informed patient consent. The authors declare that all supporting data are available within the article.
Primary murine pulmonary endothelial cells (PMECs) were isolated as we have described previously. PMECs were resuspended in diluted endothelial cell growth medium (1:3 in Dulbecco modified Eagle medium/nutrient mixture F-12) and plated on 15 mg/mL reduced-growth factor extracellular matrix (Cultrex, Trevigen) at a density of 1.75×10 5 cells/cm 2. Tubule formation was determined by measuring branch number and length by using National Institutes of Health ImageJ software, 2, 4, and 6 hours after treatment with CNP (1 nmol/L to 1 µmol/L, GenScript) or VEGF (30 ng/mL, Pre-protech).
Tubule formation in human umbilical vein endothelial cells (HUVEC) was measured after treatment with CNP (1 nmol/L) or atrial natriuretic peptide (ANP) (10 nmol/L) in the absence or presence of selective NPR-C antagonist M372049 (10 µmol/L), the selective phosphoinositide 3-kinase γ (PI3Kγ) inhibitor AS605240 (100 nmol/L; Sigma), or the selective protein kinase G (PKG) blocker KT5283 (2 μmol/L; Sigma) over a 16-hour period. A cohort of cells was transfected using lipofectamine 2000 (Thermofisher Scientific) with either nonspecific small interfering RNA (Mission Control, Sigma Aldrich), NPR-B– or NPR-C–specific small interfering RNA (Sigma Aldrich) and were treated with CNP or the selective NPR-C agonist, cANF (2 nmol/L). Silencing of NPR-B and NPR-C was confirmed at the mRNA and protein level by using reverse transcription polymerase chain reaction and immunoblot.
Endothelial cells were plated in gelatin-coated wells of a 96-well plate (Corning Biocoat) at a density of 1.75×10 5 cells/cm 2 and left to reach confluence. A scratch was performed using a 10-μL sterile pipette, and images were taken at regular intervals over a 24-hour period to monitor scratch closure. Cell populations were treated with CNP (1 nmol/L to 1 µmol/L) or VEGF (30 ng/mL).
Mice were killed by cervical dislocation, and thoracic aortas were removed, trimmed of all extraneous tissue, and flushed with media via the lumen to remove all blood. Rings of ≈0.5 mm were cut and embedded in 1 mg/mL collagen matrix (type 1 rat tail, Millipore) and incubated for 1 hour at 37°C. Opti-MEM + Glutamax media (Opti-MEM, Gibco) containing 2.5% bovine serum (Gibco), 50 U/mL penicillin, and 0.5 mg/mL streptomycin (Sigma-Aldrich) and either CNP (1 nmol/L to 1 µmol/L) or VEGF (30 ng/mL) or was added to the wells. Interventions were refreshed every 2 days and images of sprouting aortas were obtained after 7 days.
Mice were injected subcutaneously with 15 mg/mL reduced-growth factor extracellular matrix (Cultrex, Trevigen) containing 30 ng/mL VEGF (Pre-protech) and 50 U heparin (Pfizer), which formed a solid plug at body temperature. Plugs were extracted after 14 days and homogenized in 0.5 mL of cell lysis buffer and centrifuged at 6000 _g_ at 4°C for 60 minutes. Hemoglobin was detected at 400 nm wavelength by using a colorimetric assay (Sigma-Aldrich). Histological analysis of fixed and paraffin-embedded matrigel plugs was performed using hematoxylin and eosin and isolectin B4 staining.
Mice were anesthetized with 1.5% to 2% isoflurane vaporized in oxygen, placed on a heated blanket to maintain body temperature, and a small incision (≈10 mm) was made in the hindlimb skin directly over the femoral vasculature. All mice received preoperative analgesia (buprenorphine, 0.1 mg/kg, Vetergesic, Alstoe Animal health). A portion of the femoral artery was exposed via a 2-cm incision, and 2 ligations were performed using 8 nylon sutures, first distal to the origin of the profunda femoris artery and second proximal to the saphenous artery. The femoral artery was then excised between the ligation sites and the skin was closed with noncontinuous absorbable suture. Noninvasive laser Doppler imaging (Moor LDI2, Moor Instruments Ltd) was used to assess hindlimb blood flow at baseline and immediately after undergoing hindlimb ischemia (HLI), with subsequent imaging at day 3, 7, 14, 21, and 28 post-HLI. The rate of reperfusion in the hindlimb was calculated as a ratio of blood flow in the ipsilateral ischemic versus contralateral nonischemic limb. In some experiments, mice were implanted with osmotic minipumps (1002; Alzet) containing CNP (0.2 mg·kg–1·d–1) at day 0 to explore if any phenotype produced by gene knockout could be reversed by pharmacological application of the peptide. At the end of the study (day 28), mice were briefly anesthetized with isoflurane and blood was collected via cardiac puncture. Mice were culled by cervical dislocation, and the gastrocnemius muscle from each leg was harvested for postanalysis. In some experiments, the gastrocnemius muscle from each leg was harvested at day 3 and day 7 post-HLI from WT mice, treated with an RNA stabilizer (RNAlater, Sigma), and stored at –20°C before RNA isolation.
Mice were anesthetized using 1.5% to 2% isoflurane in oxygen. Left internal carot
