Inhibition of Kinin B1 Receptors Attenuates Pulmonary Hypertension and Vascular RemodelingNovelty and Significance
This study examined whether the kinin B1 receptor is involved in the pathogenesis of pulmonary hypertension, and whether its inhibition could reduce inflammation, pulmonary hypertension, vascular remodeling, and right heart dysfunction. Male Wistar rats underwent left pneumonectomy. Seven days later, the rats were injected subcutaneously with monocrotaline (60 mg/kg). The rats were then randomly assigned to receive treatment with vehicle or with BI113823 (a selective B1 receptor antagonist, 30 mg/kg, twice per day) via oral gavage from the day of monocrotaline injection to day 28. By day 28, BI113823-treated rats had significantly lower mean pulmonary artery pressure, less right ventricular hypertrophy, and pulmonary arterial neointimal formation than that of the vehicle-treated rats. Real-time polymerase chain reaction revealed that there was a significant increase in mRNA expression of B1 receptors in the lungs of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 significantly reduced macrophage recruitment, as measured via bronchoalveolar lavage. It also markedly reduced CD-68 positive macrophages and proliferating cell nuclear antigen positive cells in the perivascular areas, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase 2 and 9, and B1 receptors compared with measurements in vehicle-treated rats. These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.
- right ventricular
- vascular remodeling
- right heart hypertrophy
- kinin B1 receptor antagonist
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by persistent increases in pulmonary arterial pressure. This increasing pressure is associated with perivascular inflammation, excessive pulmonary vascular proliferation, remodeling, and vasoconstriction. Ultimately, PAH can cause right heart failure and death.1–3 Despite new treatment options, PAH patients still face high mortality rates.1–3 Therefore, it is necessary to develop additional novel therapeutic approaches that target the various components of this multifactorial disease.1–3
Kinins are proinflammatory peptides that exert a variety of biological actions via stimulation of 2 pharmacologically distinct receptor subtypes, B1 and B2.4,5 The former are normally weakly expressed, but can be upregulated in the presence of cytokines and endotoxins or during tissue injury, whereas the latter are expressed constitutively.4,5 One important difference between the 2 subtypes is that the B2 is internalized rapidly and desensitizes, whereas kinin B1 receptor–induced responses are more persistent and suggesting the lack of the internalization of the B1 receptor–ligand complex.4–6 Thus, kinin B1 receptors represent a novel therapeutic target for chronic inflammatory diseases.4–6 Kinin B1 receptors are involved in diverse pathological processes, including inflammation, smooth muscle contraction, increased vascular permeability, edema, pain, cytokine and chemokine release, cell proliferation, and vascular and myocardial remodeling.4–9 In contrast to the B2 receptor–mediated relaxation to bradykinin, B1 receptors mediate vasoconstriction in cardiopulmonary vascular beds.8,10,11 We hypothesized that kinin B1 receptors may play an important role in the pathogenesis of PAH. With this same reasoning, B1 receptor antagonists may have therapeutic potential for treating PAH by reducing inflammation, inhibiting smooth muscle contraction, attenuating vascular and cardiac remodeling, and improving cardiac function.
The small molecule BI113823 is a newly developed, orally active, nonpeptide B1 receptor antagonist that exerts a potent anti-inflammatory effect with a favorable cardiovascular profile.7,9 This study examined the effects of kinin B1 receptor blockade with BI113823 in a well-established experimental model of monocrotaline-induced PAH and vascular remodeling in left pneumonectomized rats.12
Detailed Methods are available in the online-only Data Supplement.
Monocrotaline-Induced Pulmonary Hypertension in Left Pneumonectomized Rats
On day 0, Male Wistar rats (250–300 g) were anesthetized with intramuscular injections of ketamine (80 mg/kg) and xylazine (10 mg/kg) before undergoing left pneumonectomy, as previously described by Faul et al.12 On day 7, rats were injected subcutaneously in the right hindlimb with monocrotaline (60 mg/kg, WAKO, Japan). These rats were then randomly assigned to receive treatment with vehicle (0.5% Natrosol+0.01% TWEEN 80, PO, BID, n=8) or with BI113823 (selective B1 receptor antagonist, 30 mg/kg, PO, BID, n=8) from the day of monocrotaline injection to day 28. All animals survived the entire experimental protocol.
On day 28, the right atrial pressure, right ventricular systolic blood pressure, and pulmonary arterial pressure were measured as previously described12 (for details, see the online-only Data Supplement).
Assessment of Pulmonary Inflammation
Bronchoalveolar lavage was collected and analyzed for inflammatory cell influx, total protein, tumor necrosis factor-α, and interleukin 1-β (IL-1β) levels.
Standard histopathologic procedures were used to prepare 5-μm-thick sections. The lung sections were stained with hematoxylin and eosin (Sigma, St. Louis, MO) and with Masson trichrome (American Master Tech Scientific, Inc, Lodi, CA) and were examined with light microscopy for morphological alterations.
Reverse-Transcription Polymerase Chain Reaction
Transcript levels of B1 and B2 receptors were assessed by quantitative reverse-transcription polymerase chain reaction as previously described.7
Immunohistochemical analysis for endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), CD68, proliferating cell nuclear antigen, matrix metalloproteinase (MMP)-9, MMP-2, and α-smooth muscle actin in lung sections were performed.
Western blot experiments were performed to determine protein expression of B1 receptor, eNOS, iNOS, CD68, proliferating cell nuclear antigen, MMP-9, and MMP-2 in lung tissues.
Pulmonary Vascular Function and Smooth Muscle Cell Outgrowth
Pulmonary artery relaxation responses to acetylcholine and sodium nitroprusside were measured using myograph methods. The effects of BI113823 on ex vivo pulmonary vascular smooth muscle cell migration and proliferation were performed.
All data are reported as mean±SEM. Statistical differences were determined by ANOVA for repeated measures followed by Bonferroni’s post hoc test using GraphPad Prism 5. P values <0.05 were considered statistically significant differences.
Three weeks after monocrotaline injection, compared with sham control rats, the vehicle-treated rats developed severe pulmonary hypertension with a significant increase in mean pulmonary artery pressure (↑ by 190%) and an increase in right ventricular systolic pressure (↑ by 140%; Figure 1A and 1B). While there was no significant changes in systemic blood pressure in all study groups (Table S1 in the online-only Data Supplement). As a consequence of increased pulmonary pressure, the vehicle-treated rats also developed significant right ventricular hypertrophy. The ratio of right ventricular weight/left ventricular and septum weight (RV/[LV+S]) increased by 90% in vehicle-treated rats when compared with that in sham control rats (Figure 1C). Over the course of the experiment, the vehicle-treated rats also exhibited a significant decrease in cardiac index and an increase in water content of their lungs when compared with sham control rats (Figure 1D and 1E). In contrast, treatment with BI113823 significantly reversed elevated mean pulmonary artery pressure (↓ by 52%), right ventricular systolic pressure (↓ by 39%), and RV/(LV+S) ratio (0.55 versus 0.38 in vehicle control), whereas increased cardiac index (↑ by 31%) compared with vehicle-treated rats (Figure 1A–1E). Reverse-transcription polymerase chain reaction revealed that kinin B1 and B2 receptors were expressed constitutively in rat pulmonary tissue. However, the mRNA expression of B1 receptors increased significantly in the monocrotaline-challenged pneumonectomized rats. In contrast, there was no significant change in the mRNA expression of B2 receptors in the pulmonary tissue (Figure 1F). These findings suggest that kinin B1 receptors play an important role in the development of pulmonary hypertension.
We quantitatively assessed the degree of pulmonary artery remodeling. Twenty-eight days after monocrotaline injection, the pulmonary arteries of the vehicle-treated rats were found to have extensive neointimal formation composed of α-smooth muscle actin–positive cells (Figure 2A). The pulmonary arteries from these vehicle-treated animals also had markedly increased medial wall thickness and increased vascular occlusion scores (Figure 2B and 2C). In contrast, the pulmonary arteries from animals treated with BI113823 had a significant decrease in medial wall thickness and in vascular occlusion scores. BI113823 also prevented the development of airway remodeling in rats (Figure S2). Furthermore, Masson trichrome staining showed marked decreases in collagen deposition in lung and right heart of animals treated with BI113823 compared with vehicle-treated controls (Figure 3A–3C).
Inflammatory cell recruitment is a key feature in the development of pulmonary artery hypertension. In this study, bronchoalveolar lavage samples from monocrotaline-challenged animals demonstrated a large increase in the number of macrophages and a small influx of neutrophils after monocrotaline challenge. In contrast, in rats treated with BI113823, the number of macrophages was only 40% of that of the vehicle-control animals (Figure 4A; Figure S1). Furthermore, protein content, tumor necrosis factor-α, and IL-1β levels in lavage were significantly lower in rats treated with BI113823 than those in vehicle controls (Figure 4B–4D).
Immunohistochemistry of vehicle-treated, monocrotaline-injured pulmonary tissue revealed that there was marked recruitment of CD-68 positive macrophages into perivascular areas, an increase in iNOS and a decrease in eNOS expression (Figure 5A and 5B). Immunohistochemistry also demonstrated substantial vascular cell proliferation in the thickened media layer of the PA. This layer was composed of proliferating cell nuclear antigen–positive cells in vehicle-treated monocrotaline-injured lung tissue (Figure 5C and 5D). In contrast, treatment with BI113823 attenuated the perivascular inflammation, smooth muscle cell proliferation, and nitrosative stress (Figure 5A–5D). It also restored eNOS expression (Figure 5A and 5B). Levels of MMP-2 and MMP-9 protein were also greatly increased in vehicle-treated monocrotaline-injured lung tissue. Similarly, the expression of MMP-2 and MMP-9 was strongly suppressed by BI113823 treatment (Figure 5C and 5D). The reductions of CD-68, iNOS, proliferating cell nuclear antigen, MMP-2, MMP-9, and B1 receptor expression by BI113823 were confirmed by Western blot in lung tissues, but not eNOS (Figure S3). In addition, treatment with BI113823 improved both endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to sodium nitroprusside in isolated pulmonary arteries (Figure S4) and attenuated growth factors and hypoxia-stimulated pulmonary artery smooth muscle cell migration and proliferation (Figure S5).
The kinin B1 receptor mediates various inflammatory processes. This study examined whether the kinin B1 receptor is involved in the pathogenesis of PAH, and whether B1 receptor inhibition could attenuate inflammation and vascular remodeling, therefore preventing the development of PAH. Our data showed upregulation of B1 receptor mRNA expression in lung tissues from pneumonectomized rats challenged with monocrotaline. B1 receptor inhibition (with BI113823) protected lungs from developing PAH, vascular remodeling, and right ventricular hypertrophy. Treatment with BI113823 also reduced macrophage recruitment and cytokine production, inhibited vascular cell proliferation, and reduced expression of iNOS, MMP-2, and MMP-9 proteins.
Inflammation is a prominent pathological feature in PAH.13,14 Evidence from studies in animal models and in patients with pulmonary hypertension suggests that inflammation contributes to the development of PAH.12–16 In lung biopsies from patients with PAH, infiltration of inflammatory cells, including macrophages and T and B lymphocytes, and dendritic cells is found in pulmonary perivascular spaces and around the plexiform lesions in PAH.17,18 Activation of macrophages induces the release of cytokines, such as tumor necrosis factor-α, IL-1β, and IL-6, which all are major contributing factors in the pathogenesis of PAH.19,20 In this study, bronchoalveolar lavage samples from monocrotaline-challenged left pneumonectomized rats demonstrated a large increase in the number of macrophages and a small influx of neutrophils after monocrotaline challenge. Furthermore, there was marked recruitment of CD-68 positive macrophages into perivascular areas of vehicle-treated, monocrotaline-injured pulmonary tissues. These findings further support an important role of inflammation in the pathogenesis of PAH, and that control of inflammation could be important for the prevention or treatment of PAH.
Kinin B1 receptors become upregulated following proinflammatory stimuli and then mediate diverse pathological processes.4–6 In patients with atheromatous disease, there is high kinin B1 receptor expression, but low kinin B2 receptor expression on foamy macrophages within thickened intimal plaques.21 Activation of B1 receptors stimulate leukocyte activation and chemotaxis, synthesis of cytokines and chemokines, and also increase vascular permeability.3,4,10 In this study, B1 receptor expression was upregulated in the lung tissues of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 reduced the number of macrophages infiltration in bronchoalveolar lavage and the CD-68 positive macrophages into perivascular areas. Furthermore, the reduction in macrophage infiltration was accompanied by a significant reduction in proinflammatory cytokine tumor necrosis factor-α, IL-1β release, and B1 receptor expression. These finding suggests that the BI113823-induced inhibition of the PAH and vascular remodeling is attributed to inhibition of macrophage recruitment and cytokine production.
Pulmonary vascular cell proliferation and vascular remodeling is another important feature of PAH.1,2 In the media, smooth muscle cell proliferation, resulting in thickening of the vessel wall and muscularization of the arterioles, leads to both stiffening and vasoconstriction of the vessels.22 The increased pulmonary vascular resistance will consequently lead to right ventricular hypertrophy and dysfunction and premature death.1,2,22 Inflammatory processes play an important role in the cardiopulmonary remodeling of PAH.13–16 The degree of perivascular inflammation correlates with both vascular wall thickness and the mean pulmonary arterial pressure.18 Cytokines/chemokines and hypoxia are key players in the pathogenesis of PAH by mediating the excess cellular proliferation and pulmonary vascular remodeling.13–16 Human atherosclerotic lesions express high level of B1 receptors.21 Kinin B1 receptors mediate vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue in mice and rats.23 In this study, BI113823 significantly inhibited both chemokines- and hypoxia-induced migration and proliferation of rat pulmonary artery smooth muscle cells from pulmonary arterial explants. Furthermore, treatment with BI113823 inhibited the perivascular macrophages recruitment, reduced expression of iNOS, MMP-2, and MMP-9 proteins, and reduced pulmonary and cardiac fibrosis. Collectively, these effects prevented the development of PAH, vascular remodeling, and right ventricular hypertrophy.
Another key feature of PAH is vasoconstriction because of constrictive agents, such as endothelin-1, and an imbalance of vasodilators and vascular remodeling.22,24 Recent studies found little or no expression of NOS in the pulmonary vascular endothelium of patients with pulmonary hypertension.25 Various mediators, including cytokines, endotoxin, angiotensin II, and endothelin-1, induce transcriptional upregulation of kinin B1 receptors in vascular smooth muscle cells and subsequently increase vasoconstriction in cardiopulmonary vascular beds.8,10,11 B1 receptors have been shown to mediate strong contractile response to B1 receptor agonist des-Arg9-BK in pulmonary arteries in neonatal group B streptococcal sepsis in piglets and likely participate in the increase of pulmonary vascular resistance.10 Des-Arg9-BK decreases epicardial diameters in heart transplant patients.11 In isolated pig coronary arteries, we found that B1 receptors mediate strong, endothelium-independent coronary constriction in endotoxin-damaged arteries, but has no response in normal coronary arteries.8 Furthermore, B1 receptors mediate vasoconstriction in cardiopulmonary vascular beds by coupling to cyclooxygenases-2 and activation of thromboxane-prostanoid receptors.8,10 Kinin production increases during inflammation and inflammatory cells are sources of tissue kallikrein.26 Therefore, it is logical to presume that the local kinin production during inflammation can have pathological significance in the pulmonary vascular tone and leading to PAH. In addition, endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to sodium nitroprusside were impaired in vehicle-treated, monocrotaline-injured pulmonary arteries in this study. In contrast, treatment with BI113823 improved both endothelium-dependent and endothelium-independent relaxations in pulmonary arteries. Therefore, in addition to inhibition of inflammation and cardiopulmonary remodeling, kinin B1 receptor blockade may protect from PAH by regulating pulmonary vascular tone.
BI113823 is a small molecule orally active, nonpeptide B1 receptor antagonist that exerts a potent anti-inflammatory effect with a favorable cardiovascular profile.7,27 It exhibits high affinity (Ki) for both human and rat B1 receptors (5.3 and 13.3 nmol/L, respectively), whereas has no affinity for the B2 receptors (IC50>10.000 nmol/L).27 BI 113823 does not influence blood pressure in conscious rats. Furthermore, BI 113823 does not interfere with the blood pressure–lowering effects of lisinopril in spontaneously hypertensive rats after a 2-week treatment period.27 This study examined the effects of BI113823 in PAH in an experimental model of monocrotaline-induced PAH in pneumonectomized rats. This method produced severe pulmonary hypertension, right ventricular hypertrophy, and formation of obstructive intimal lesions in the peripheral pulmonary arterials.12 However, this animal model may not precisely simulate all the forms of the pathobiology that might be present clinically in patients with idiopathic and heritable PAH.
Data from this study indicate that kinin B1 receptors play distinct roles in the development of PAH and vascular remodeling in an experimental rat model. Kinin B1 receptor inhibition may provide multiple protective factors against PAH, including reduction of inflammation and cytokine production, inhibition of smooth muscle constriction, reduction of vascular cell proliferation and vascular remodeling, and attenuation of right heart hypertrophy. Further studies are warranted to further elucidate the therapeutic potential of kinin B1 receptor antagonists for PAH.
Sources of Funding
This work was supported in part by Boehringer Ingelheim Pharma GmbH & Co. KG; the Brain Korea 21 PLUS Project, National Research Foundation; and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2012007331), Korea.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.115.05338/-/DC1.
- Received February 10, 2015.
- Revision received March 1, 2015.
- Accepted August 4, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is New?
Data from this study reveal that kinin B1 receptors play an important role in the pathogenesis of pulmonary arterial hypertension.
What Is Relevant?
We report that the newly developed, small molecule, orally active, nonpeptide B1 receptor antagonist, BI113823 attenuated pulmonary arterial hypertension, vascular remodeling, and right ventricular hypertrophy. Treatment with BI113823 also reduced macrophage recruitment and cytokine production, inhibited vascular cell proliferation, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 proteins, and enhanced endothelial nitric oxide synthase expression.
These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.