Targeting Kinin B1 Receptor for Therapeutic Neovascularization
Background— Kinins are modulators of cardiovascular function. After ischemic injury, enhanced kinin generation may contribute in processes responsible for tissue healing.
Methods and Results— Using pharmacological and genetic approaches, we investigated the role of kinin B1 receptor in reparative angiogenesis in a murine model of limb ischemia. The effect of B1 pharmacological manipulation on human endothelial cell proliferation and apoptosis was also studied in vitro. Abrogation of B1 signaling dramatically inhibited the native angiogenic response to ischemia, severely compromising blood perfusion recovery. Outcome was especially impaired in B1 knockouts that showed a very high incidence of limb necrosis, eventually leading to spontaneous auto-amputation. Conversely, local delivery of a long-acting B1 receptor agonist enhanced collateral vascular growth in ischemic skeletal muscle, accelerated the rate of perfusion recovery, and improved limb salvage. In vitro, B1 activation stimulated endothelial cell proliferation and survival, whereas B1 antagonism induced apoptosis.
Conclusions— Our results indicate that the B1 plays an essential role in the host defense response to ischemic injury. B1 signaling potentiation might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.
Received July 12, 2001; revision received October 31, 2001; accepted November 2, 2001.
Ischemic disease represents a major health problem and is expected to become the new worldwide epidemic of the third millennium. After occurrence of vascular occlusion, clinical outcome mainly depends on the native potential to develop new collaterals, a defense response aimed to maintain tissue perfusion and function. However, under certain conditions such as diabetes, hypercholesterolemia, and hypertension, post-ischemic healing is compromised because of a defective modulation of angiogenic growth factors.1–3⇓⇓ Thus, supplementation with angiogenic substances has been proposed as a possible mechanism to overcome the failure in mounting an adequate vascular response.4,5⇓
The kallikrein-kinin system (KKS), long regarded as a modulator of cardiovascular homeostasis, nociception, and inflammation,6,7⇓ is now being considered for its potential in reparative and therapeutic angiogenesis.3,8,9⇓⇓ Kinin peptides cleaved from kininogens by kallikreins interact with G protein-coupled B1 and B2 receptors (B1 and B2), thus leading vascular endothelial cells (ECs) to release autacoids such as nitric oxide (NO) and prostaglandins.10,11⇓ The constitutive B2 is responsible for the majority of biological effects of bradykinin (BK) and Lys-BK. In contrast, the B1 preferentially binds desArg9-BK and Lys-desArg10-BK, is induced by tissue damage via activation of transcription factor nuclear factor κB, and does not desensitize after exposure to ligand.12–14⇓⇓ Circumstantial evidence for a role of B1 in reparative angiogenesis comes from the observation that B1 gene expression is up-regulated by ischemia in skeletal muscle or myocardium.8,15⇓ In vitro, kinins promote EC proliferation via activation of the B1-cAMP pathway.16,17⇓ In vivo, B1 stimulation induces neovascularization in the rabbit cornea17 and is responsible for capillary proliferation evoked by BK18 or tissue kallikrein gene transfer.8,19⇓
The aim of the present study was to firmly establish the functional relevance of B1 in post-ischemic neovascularization. We demonstrated that B1 signaling plays an essential role in reparative angiogenesis by modulating EC proliferation and survival. Moreover, potentiation of this mechanism exerts a therapeutic effect by accelerating spontaneous healing. Exploitation of the angiogenic potential of B1 agonists may be envisaged for the treatment of patients with peripheral artery disease.
All procedures complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Md, 1996). CD1 mice were purchased from Charles River, Comerio, Italy. B1 knock outs20 were provided by Max Delbrück Center for Molecular Medicine (Berlin-Buch, Germany), whereas their wild-type J129 controls were obtained from Jackson Laboratories (Bar Harbor, Me).
In Vivo Experimental Protocols
With mice under the effect of 2,2,2 tribromoethanol anesthesia (880 mmol/kg BW IP, Sigma-Aldrich), the left femoral artery was exposed, dissected free, and excised.8 Three groups were studied: (1) CD1 mice were given the B1 antagonists desArg9-[Leu8]-BK (DALBK from Sigma-Aldrich, at 50 nmol/kg BW per day, n=9) or AcLys[D-βNal7,Ile8]-desArg9-BK (R715, at 50 nmol/kg BW per day, n=7), or vehicle (sterile saline, n=8), and were followed-up for 21 days from surgery. Drugs were delivered IP by osmotic mini-pumps (Alza Co., Palo Alto, CA). Selectivity of antagonists has been previously reported.7 (2) The second group consisted of B1 knockouts (n=15) and J129 controls. The latter were subdivided into 2 groups given DALBK (n=8) or saline (n=10) IP. Mice were euthanized 14 days from surgery. (3) CD1 and J129 mice had their left adductor muscles injected with B1 agonist Sar[D-Phe8]des-Arg9-BK (R916, at 2.5 μmol/kg BW in 20 μL saline once or twice a day, starting on the day of surgery, n=10 each dose per strain) or vehicle (n=10 each strain) and were followed-up for 14 days from ischemia.
Histological Assessment of Neovascularization
Capillary and myofiber densities of both adductors were determined at necroscopy. Anesthetized mice were perfused with PBS (1 minute) and then with 10% buffered formalin (10 minutes) at 100 mm Hg via the abdominal aorta. After paraffin embedding, 3 μm-thick sections were cut from each sample with muscle fibers transversely oriented, and stained with hematoxylin and eosin.
Sections were analyzed in a blinded fashion using an ocular reticle (9604-μm2 area) at 1000× magnification. Twenty-five fields were randomly counted and averaged. The number of capillary (ncap) and myofiber (nfiber) profiles per field was used to compute capillary or myofiber numerical density per square mm of muscle, according to the following formulas: ncap/mm2 = ncap in total fields/total field area and nfiber/mm2 = nfiber in total fields/total field area. In addition, capillary density was normalized to myofiber density (ncap/nfiber).3,8⇓
Post-Ischemic Hemodynamic Recovery
Hindlimb blood flow of anesthetized mice was measured by laser Doppler flowmetry (Lisca, Sweden) under basal conditions and then weekly after surgery, and the ratio of perfusion between ischemic to non-ischemic foot was calculated.8
EC Proliferation and Apoptosis
Human umbilical vein ECs (HUVECs) were cultured in M199 supplemented with 20% fetal bovine serum (FBS, Gibco), 0.3% bovine brain extract (BBE, Clonetics), and antibiotics, and were maintained at 37°C in 5% CO2/95% air. Experiments were performed on cells at passages between 2 and 5.
The effect of B1 stimulation on HUVEC proliferation was assayed after a total of 24 hours of serum starvation. In a set of experiments, the last 2 hours of starvation were combined with incubation in a modified Krebs buffer (137 mmol/L NaCl, 3.8 mmol/L KCl, 0.49 mmol/L MgCl2, 0.9 mmol/L CaCl2, and 4.0 mmol/L Hepes) supplemented with 10 mmol/L 2-deoxyglucose, 20 mmol/L sodium lactate, 1 mmol/L sodium dithionite, and 12 mmol/L KCl (pH 6.5) to evoke simulated ischemia (SI).21 Then, ischemic or not ischemic HUVECs were incubated for 72 hours in M199 medium containing 1% FCS and added with the B1 agonist R916 (50 nmol/L to 1 μmol/L) or vehicle (PBS). In a separate experiment, R916 (100 nmol/L) was co-incubated with either R715 (1 μmol/L) or PBS. Incubation was carried out in the presence of 1 μCi/mL [3H] thymidine (specific activity 5 Ci/mmol, Amersham Pharmacia Biotech). The medium was then removed and the cell monolayer in each well was washed twice with PBS, exposed to 5% trichloroacetic acid (500 μL) for 5 minutes, and fixed in methanol (500 μL). Finally, cells were digested by adding 25 mol/L formic acid (500 μL). Each digest was transferred to a scintillation vial and radioactivity was determined by a scintillation counter (LKB Instrument Inc). Experiments were repeated at least 6 times.
To assess the effect of B1 blockade on apoptosis, HUVECs suspended in a normal culture medium were seeded in gelatinated 24-well dishes at a density of 4×104 cells per well, and allowed to attach overnight. Fresh media supplemented with low (2%) or normal (20%) FBS and containing R916 (100 nmol/L to 5 μmol/L), R715 (100 nmol/L to 5 μmol/L), or PBS was then added for 24 or 48 hours. Experiments were performed at the standard atmosphere or under hypoxia obtained by placing cell plates in a modular incubator chamber (Billups-Rothenberg) filled with 5%CO2/95%N2. At the end of the incubation period, supernatant was collected for LDH determination by the Promega Cytotoxic Assay and apoptosis was investigated by 4′-6′-diaminidino phenylindole (DAPI) staining (Roche). To this aim, cells were fixed in 4% paraformaldehyde for 10 minutes, washed twice with PBS and incubated with DAPI (1 μg/mL PBS) for 20 minutes. Wells were examined by fluorescent microscopy at 100× magnifications. Three randomly selected fields per well were photographed with a videocamera and then analyzed for total cell number. Apoptotic HUVECs, identified by condensation and fragmentation of their nuclei, were counted by a researcher blinded to the treatment. In each examined field, the total cell number was also computed to calculate the percent of apoptotic cells. Each experiment was repeated 3 to 6 times.
All results are expressed as mean±SEM. Multivariate repeated-measures ANOVA was performed to test for interaction between time and grouping factor. In multiple comparisons among independent groups in which ANOVA and F test indicated significant differences, the statistical value was determined according to the Bonferroni’s method. Differences within and between groups were determined using paired or unpaired Student’s t test, respectively. The comparative incidence of limb salvage was evaluated by χ2 analysis. A P value <0.05 was interpreted to denote statistical significance.
Impact of B1 Signaling on Microvascular Response to Ischemia
As depicted in Figure 1A, vehicle-treated J129 showed a 1.7-fold increase in adductor muscle capillarity 14 days from ischemia induction (950±158 versus 544±66 cap/mm2 in contralateral normoperfused muscles, P<0.01). The reparative response was attenuated by DALBK (773±71 versus 603±92 cap/mm2 in contralateral muscles, P=NS) and absent in B1 knockouts (367±59 versus 383±45 cap/mm2 in contralateral muscles, P=NS). Results did not differ when expressed as capillary to myofiber ratio (Figure 1B).
As shown in Figure 2, the instrumental relevance of endogenous B1 signaling was confirmed in the CD1 strain. In fact, although capillary density was increased by 1.9-fold 3 weeks from induction of ischemia in vehicle-treated animals (1008±137 versus 516±79 cap/mm2 in contralateral adductors, P<0.001), this response was prevented by either DALBK or R715 (P<0.01 for both comparisons). Similar results were obtained after normalization of capillarity by fiber number (data not shown).
We then evaluated whether reparative neovascularization would be potentiated by R916 agonist local administration. Because vascular effects of the 2 doses tested were superimposable, results were cumulated. As shown in Figure 3A, capillary density of ischemic adductors was enhanced in R916-treated CD1 (1207±46 versus 917±112 cap/mm2 in vehicle group, P<0.005). Results were comparable when expressed as a capillary-to-myofiber ratio (Figure 3B). In contrast, capillarity of contralateral normoperfused non-injected muscles remained unaffected. Similar results were obtained in R916-treated J129, whereas, as expected, the agonist did not improve the deficient capillary response typical of B1 knockouts (data not shown).
Effect of B1 Manipulation on Post-Ischemic Perfusion Recovery
As depicted in Figure 4, CD1 showed an impaired hemodynamic recovery under chronic B1 blockade by either R715 or DALBK. In fact, although 3 weeks were enough for vehicle-treated mice to recover, the limbs of antagonist-treated mice were still hypoperfused at the end of this time frame (P<0.01).
As shown in Figure 5, recovery of mice with B1 genetic disruption was dramatically compromised, due to the virtually absent neovascularization (see above). Limb necrosis developed in the majority of B1 knockouts, leading to spontaneous auto-amputation in 13 out 15 animals (87 versus 29% in J129, P<0.01). The 2 remainders showed persistent limb ischemia at 2 weeks from surgery, with a blood perfusion to the ischemic limb 69 and 63% less than that of contralateral side. In consideration of the severe outcome, animals were sacrificed at this stage to avoid unnecessary pain.
Conversely, local injection of the B1 agonist R916 into the ischemic muscles of J129 improved clinical outcome (P<0.001). Limb salvage occurred within 2 weeks in 12 (60%) out 20 mice. Among remainders, 3 (15%) showed residual ischemia and 5 (25%) experienced foot necrosis. As shown in Figure 6, sequential analysis of limb blood flow denoted an accelerated recovery in R916-treated groups. With the highest dose, a beneficial effect was already detected at 1 week.
Effect of B1 Signaling on EC Proliferation and Survival
Pharmacological manipulation of the B1 affects HUVEC proliferation and apoptosis. After SI preconditioning, HUVEC spontaneous mitotic activity was higher (data not shown, P<0.05) and the response to R916 increased (2.1-fold versus 1.5-fold in not SI in response to 100 nmol/L R916, P<0.05), suggesting that the B1 receptor might be up-regulated by SI. Figure 7 shows the dose-response to R916 after SI. The proliferative effect of R916 was prevented by R715 (Figure 7), whereas R715 per se was ineffective (data not shown).
In experiments performed at low serum concentration, R916 reduced apoptosis (Figure 8A and 8B), whereas R715 had no effect (data not shown). On the other hand, R715 increased apoptosis of HUVECs cultured under standard FBS concentration (Figure 8C and 8D), suggesting that B1 ligands generated by ECs from serum-derived substrates may act autocrinally to favor cell survival.22 LDH levels did not differ among groups (data not shown), excluding a cytotoxic effect of R715. No differences were seen when cells were incubated under hypoxic conditions (data not shown).
Circumstantial evidence derived from pharmacological studies suggests a role for the B1 receptor in capillary proliferation.8,16–19⇓⇓⇓⇓ By exploiting the powerful tool of gene knockout strategy, we demonstrate that the endogenous B1 signaling is indeed essential for developing new blood vessels with functional relevance for limb post-ischemic recovery. Another important finding of the present study is the discovery that B1 stimulation by local administration of a specific receptor agonist has a potential in therapeutic angiogenesis.
Increased kinin levels in the coronary sinus blood of dogs and humans have been reported after myocardial ischemia.23,24⇓ For a long time, the compensatory relevance of this phenomenon has been attributed to the ability of kinins to cause vasodilation and preserve muscular energy content.25,26,27⇓⇓ More recently, the theory has been proposed that kinins may exert long-term protection on ischemic tissue by potentiation of neovascularization. We and others have documented that B1 gene expression is activated in ischemic skeletal muscles and myocardium.8,15⇓ Furthermore, in vitro studies showed that engagement of kinin B1 by des-Arg10-kallidin or R916 stimulates proliferation of coronary ECs.16,17⇓ We found that the mild proliferative effect of R916 on HUVECs under normal conditions is potentiated by preconditioning with a culture system mimicking an ischemic milieu. This finding favors possible implication of the B1 in the EC program leading to capillarization in response to an ischemic injury. Furthermore, we demonstrated that the B1 plays a role in EC survival.
Seminal studies from our group showed that B1 pharmacological blockade inhibits capillary proliferation in response to limb ischemia.8 This assumption was confirmed by the use of 2 different antagonists and replicated in both wild-type J129 and CD1 strains. Moreover, we provided the new information that B1 antagonism severely impairs blood perfusion recovery of ischemic muscles. Another major novelty of the present study is the observation that genetic disruption of the B1 results in the absolute failure to mount reparative angiogenesis. The vascular deficit led to an unusual incidence of limb necrosis and auto-amputation. Congenital absence of the receptor could account for the severe outcome of B1 knockouts compared with that of antagonist-treated animals. Moreover, the mild reduction in capillarity that was evidenced in not ischemic limbs of knockouts calls for further studies aimed to assess a potential role of B1 in vasculogenesis.
Besides acting as direct modulators of EC growth and survival, kinins may participate in post-ischemic neovascularization by increasing extravasation of plasma proteins that would provide a provisional scaffold for growing vasculature. Furthermore, kinins are potent attractants for leukocytes, which represent an additional source of EC growth factors, as well as kallikrein and kininogen, ie, the components required for kinin generation.6,28⇓ Interestingly enough, B1 knockouts feature a drastic reduction in the extravasation of leukocytes in inflamed tissues,20 a deficit that may eventually contribute in impaired neovascularization.
Another intriguing possibility regards coupling of B1 receptor to neuronal modulation of angiogenesis, as supported by the observation that kinin challenge stimulates non-adrenergic non-cholinergic C-fibers to release the pro-angiogenic tachykinin neuropeptides.29 Accordingly, the alteration in C-fiber trafficking that has been reported in B1 knockouts20 may contribute in impaired neovascularization.
The present study clearly indicates that ligands activating the B1 possess a significant therapeutic potential for the treatment of peripheral ischemia. Interestingly, R916 also increases capillarity of normoperfused muscle (Costanza Emanueli, unpublished results, 2001), suggesting a potential application in patients experiencing intermittent ischemia. Administration of B1 agonists could be safer than adenovirus-mediated growth factor gene delivery that reportedly produces harmful side effects mainly attributed to viral vectors. Furthermore, receptor agonists are devoid of immunogenic activity. This fact represents another important advantage over adenovirus-mediated gene therapy, whose immunogenicity precludes long-term duration of transgene expression as well as effectiveness in case of readministration.
It could be argued that uncontrolled B1 activation might trigger local or systemic inflammatory reactions in susceptible individuals. However, such a strategy is made less risky by the availability of potent receptor antagonists. Furthermore, because of their ability to interfere with the angiogenic program, B1 receptor antagonists might be envisaged not only as kinin antidotes but also as therapeutic reagents to combat the pathological angiogenesis in cancer and chronic inflammatory diseases. Thus, B1 pharmacological manipulation might open new avenues for the treatment of a wide range of diseases characterized by impaired or excessive vascular growth.
This work was supported by a grant from the Juvenile Diabetes Research Foundation (JDRF, no. 2000/49), USA to Dr Madeddu and by a PROBRAL grant of the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and the German Academic Exchange Service to Dr Bader and Dr Pesquero. During a limited period of the study, Dr Emanueli was a visiting scientist at the Cardiovascular Research Laboratory of the St Elizabeth’s Medical Center of Boston, Mass, sponsored by the Italian Society of Cardiology. Dr Cayla is a recipient of a MDC fellowship. The support of Dr Dirk Walter for counting apoptosis is acknowledged.
- ↵Couffinhal T, Silver M, Kearney M, et al. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE −/−mice. Circulation. 1999; 99: 3188–3198.
- ↵Emanueli C, Salis MB, Stacca T, et al. Rescue of impaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue kallikrein gene transfer. Hypertension. 2001; 38: 136–141.
- ↵Baumgartner I, Pieczek A, Manor O, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation. 1998; 97: 1114–1123.
- ↵Madeddu P, Varoni MV, Palomba D, et al. Cardiovascular phenotype of a mouse strain with disruption of B2 receptor gene. Circulation. 1997; 96: 3570–3578.
- ↵Emanueli C, Minasi A, Zacheo A, et al. Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia. Circulation. 2001; 103: 125–132.
- ↵Ni A, Chao J, Chao L. Transcription factor nuclear factor kappa B regulates the inducible expression of the human B1 receptor gene in inflammation. J Biol Chem. 1998; 273: 2784–2791.
- ↵Parenti A, Morbidelli L, Ledda F, et al. The bradykinin/B1 receptor promotes angiogenesis by up-regulation of endogenous FGF-2 in endothelium via the nitric oxide synthase pathway. FASEB J. 2001; 2001: 15: 1487–1489.
- ↵Emanueli C, Zacheo A, Minasi A, et al. Adenovirus-mediated human tissue kallikrein gene delivery induces angiogenesis in normoperfused skeletal muscle. Arterioscler Thromb Vasc Biol. 2000; 20: 2364–2370.
- ↵Pesquero JB, Araujo RC, Heppenstall PA, et al. Hypoalgesia and altered inflammatory responses in mice lacking B1 receptors. Proc Natl Acad Sci U S A. 2000; 97: 8140–8145.
- ↵Ladilov Y, Schafer C, Held A, et al. Mechanism of Ca(2+) overload in endothelial cells exposed to simulated ischemia. Cardiovasc Res. 2000; 47: 394–403.
- ↵Nishikawa K, Shibayama Y, Kuna P, et al. Generation of vasoactive peptide bradykinin from human humbelical vein endothelium-bound high molecular weight kininogen by plasma kallikrein. Blood. 1992; 80: 1980–1988.
- ↵Emanueli C, Maestri R, Corradi D, et al. Dilated and failing cardiomyopathy in bradykinin B2 receptor knockout mice. Circulation. 1999; 100: 2359–2365.
- ↵Linz W, Wiemer G, Gohlke P, et al. Contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev. 1995; 47: 25–49.
- ↵Mayfield RK, Shimojo N, Jaffa AA. Skeletal muscle kallikrein: potential role in metabolic regulation. Diabetes. 1996; 45: S20–S30.
- ↵Wohlfart P, Dedio J, Wirth K, et al. Different B1 kinin receptor expression and pharmacology in endothelial cells of different origin and speciments. J Pharmacol Exp Ther. 1997; 280: 1109–1116.
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