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. Author manuscript; available in PMC: 2009 Jun 22.
Published in final edited form as: Hypertension. 2008 Sep 2;52(4):715–720. doi: 10.1161/HYPERTENSIONAHA.108.114587

Tissue Kallikrein Elicits Cardioprotection by Direct Kinin B2 Receptor Activation Independent of Kinin Formation

Julie Chao *, Hang Yin *, Lin Gao *, Makoto Hagiwara *, Bo Shen *, Zhi-Rong Yang *, Lee Chao *
PMCID: PMC2699749  NIHMSID: NIHMS118673  PMID: 18768400

Abstract

Tissue kallikrein exerts various biological functions through kinin formation with subsequent kinin B2 receptor activation. Recent studies showed that tissue kallikrein directly activates kinin B2 receptor in cultured cells expressing human kinin B2 receptor. In the present study, we investigated the role of tissue kallikrein in protection against cardiac injury through direct kinin B2 receptor activation using kininogen-deficient Brown Norway Katholiek (BNK) rats after acute myocardial infarction (MI). Tissue kallikrein was injected locally into the myocardium of BNK rats after coronary artery ligation, with and without co-injection of icatibant (a kinin B2 receptor antagonist) and L-NAME (a nitric oxide synthase inhibitor). One day after MI, tissue kallikrein treatment significantly improved cardiac contractility, reduced myocardial infarct size and left ventricle-end-diastolic pressure in BNK rats. Kallikrein attenuated ischemia-induced apoptosis and monocyte/macrophage accumulation in the ischemic myocardium in conjunction with increased nitric oxide (NO) levels and reduced myeloperoxidase activity. Icatibant and L-NAME abolished kallikrein’s effects, indicating a kinin B2 receptor-NO-mediated event. Moreover, inactive kallikrein had no beneficial effects in cardiac function, myocardial infarction, apoptosis or inflammatory cell infiltration after MI. In primary cardiomyocytes derived from BNK rats under serum-free conditions, active, but not inactive, kallikrein reduced hypoxia/reoxygenation-induced apoptosis and caspase-3 activity, and the effects were mediated by kinin B2 receptor/NO formation. This is the first study to demonstrate that tissue kallikrein directly activates kinin B2 receptor in the absence of kininogen to reduce infarct size, apoptosis and inflammation and improve cardiac performance of infarcted hearts.

Keywords: tissue kallikrein, kinin B2 receptor, cardiac function, infarct size, apoptosis

Introduction

Tissue kallikrein is a serine proteinase that specifically processes low molecular weight kininogen to produce the potent vasoactive kinin peptides bradykinin (BK) and Lys-BK (kallidin),1 which bind to and activate the kinin B2 receptor.2 Kinins have been shown to protect against cardiac injury through kinin B2 receptor activation.3,4 Yang and coworkers5 demonstrated that the cardioprotective effect of preconditioning was abolished in kinin B2 receptor knockout mice and in kininogen-deficient rats. The cardioprotective response to inhibition of angiotensin converting enzyme (ACE) and angiotensin (Ang) II type 1 receptor was diminished in B2 receptor-deficient mice.6 Similarly, kinins appear to play an important role in the cardioprotective effect of ACE inhibition in kininogen-deficient rats.7 However, recent studies from Erdös’ group demonstrated that tissue kallikrein directly activates the kinin B2 receptor in cultured CHO cells.8,9 This novel finding is consistent with our earlier report that tissue kallikrein directly induced rat uterine contraction independent of detectable kinin formation.10 In contrast, contraction of isolated jugular vein by tissue kallikrein appears to be dependent on blood vessel-derived kininogen and B2 receptor activation.11 Taken together, these results indicate that tissue kallikrein’s actions are mediated by kinin B2 receptor activation with or without kinin formation. However, whether tissue kallikrein can directly act on the kinin B2 receptor in the absence of kinin formation in triggering cardioprotective effects in vivo has not been demonstrated.

Brown Norway Katholiek (BNK) rats, genetically deficient in kininogen secretion, retain kinin B2 receptor expression. Therefore, the kininogen-deficient BNK rat is an ideal model to investigate whether tissue kallikrein has a direct effect on kinin B2 receptor in triggering biological functions. In this study, we determined whether tissue kallikrein has a cardioprotective role by direct activation of the kinin B2 receptor in BNK rats after myocardial infarction (MI). Our results showed that tissue kallikrein through kinin B2 receptor activation and nitric oxide (NO) formation improved cardiac performance and reduced ischemia-induced infarction, cardiomyocyte apoptosis and intramyocardial inflammation in BNK rats. This is the first study to identify a direct biological function of tissue kallikrein via kinin B2 receptor activation independent of kinin formation in vivo.

Methods

Animals and Treatments

Brown Norway Katholiek (BNK) rats were kindly provided by Dr. Ed Shesely of Hypertension and Vascular Research Division, Henry Ford Hospital (Detroit, Michigan) and were initially obtained from the Department of Pharmacology, Kitasato University School of Medicine. Male BNK rats weighing 200 to 250 g were subjected to ligation of the left coronary artery as previously described.12 This study complied with the Guides for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences). Rat tissue kallikrein was purified and characterized as previously described.13 Animals were randomly divided into six groups (n=10 in each group). In two control groups, rats were subjected to either sham surgery or left anterior descending (LAD) coronary artery ligation followed by saline injection. In the third group, tissue kallikrein (TK, 25 µg in 150 µl saline) was injected at 7 different sites into the border area of the infarcted left ventricle, immediately after coronary artery ligation. The fourth group received TK together with co-injection of the kinin B2 receptor antagonist (icatibant, 15 µg/rat; obtained from Hoechst Marion Roussel). The fifth group received TK followed by intravenous injection of L-NAME (35 mg/kg). The sixth group received inactive TK in a similar manner. One day after coronary artery ligation, hemodynamic parameters were analyzed as previously described,14 animals were euthanized, and heart tissues were harvested for morphological and biochemical analyses.

Inactivation of Tissue Kallikrein by Aprotinin

TK was inactivated via prior incubation with 5-fold molar excess of aprotinin at 37°C for 1 hour. Inactivation of tissue kallikrein was determined by enzymatic assay with S2266, a chromogenic substrate.15 Aprotinin-treated tissue kallikrein exhibited less than 5% of active kallikrein activity.

Myocardial Infarct Size Determination

The middle part of the heart (2 mm) was sectioned transversely and incubated with 1.5% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) for 5 minutes at 37°C. The ratio of infarcted area to the area at risk was then calculated. The infarcted area was distinguished by TTC staining using computer-assisted planimetry (NIH Image 1.57).

Histological and Immunohistological Analysis

For histological analyses, the left ventricle was fixed with 4% paraformaldehyde, dehydrated, embedded and cut into 4-µm sections. Primary antibody against ED-1 (Chemicon, 1:200) was used for immunostaining of monocytes/macrophages. The number of ED-1-positive cells was counted in a double-blind fashion from 8–10 different fields of each section (n=10) at 400 × magnification. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).16 The ratio of TUNEL-positive cardiomyocytes to the total number of cardiomyocytes was calculated.

Hypoxia/Reoxygenation (H/R) of Primary Cultured Cardiomyocytes

Cardiomyocytes were isolated from the hearts of 2- to 3-day-old BNK rats as previously described.17 Cardiomyocytes were grown in DMEM, supplemented with 10% fetal bovine serum. Cardiomyocyte origin was confirmed immunocytochemically using antibody to sarcomeric α-actinin (Sigma). Subcultured cells were maintained in serum-free DMEM for 24 hours and then incubated with serum-free DMEM supplemented with active tissue kallikrein (0.2 µM) or aprotinin-inactivated tissue kallikrein (0.2 µM) under the condition of hypoxia for 12 hours (95% N2 and 5% CO2) followed by 24-hour reoxygenation (95% O2 and 5% CO2). Apoptotic cardiomyocytes were identified by Hoechst 33342 staining. Hoechst-positive apoptotic cells were determined by counting cardiomyocytes in 6 randomly chosen fields. Caspase-3 activity in cardiomyocyte lysates was determined using a fluorometric caspase-3 assay kit (Oncogene) according to the manufacturer’s instructions.

Nitrate/Nitrite and Myeloperoxidase Assays

Nitrate/nitrite (NOx) levels, an indicator of NO production, were measured by a fluorometric assay as previously described.18 Myeloperoxidase activity in cardiac extracts was measured as previously described.19

Statistical Analysis

Data were compared among experimental groups using ANOVA followed by Fisher’s PLSD. Data are expressed as mean ± SEM. Differences were considered statistically significant at a value of P<0.05.

Results

Kallikrein Improves Cardiac Function and Reduces Infarct Size in Kininogen-Deficient Rats

Tissue kallikrein injection significantly improved cardiac function in BNK rats 1 day after MI (Table 1). MI induced a significant increase of LVEDP compared to the sham group, whereas kallikrein significantly reduced LVEDP. Cardiac contractility (dP/dt max and dP/dt min) was markedly reduced after MI, but was significantly increased by kallikrein. Both icatibant and L-NAME blocked kallikrein’s cardioprotective effects. Unlike active kallikrein, aprotinin-inactivated kallikrein did not improve cardiac contractility or reduce LVEDP in BNK rats after MI. MAP was not altered among all groups.

Table 1.

Hemodynamic Parameters 1 Day after Myocardial Infarction

MI
Parameter Sham Control TK TK/Icatibant TK/L-NAME Inactive TK
MAP (mmHg) 105.8 ± 3.6 108.3 ± 3.7 109.2 ± 2.5 108.3 ± 4.1 104.2 ± 3.5 106 ± 4.1
LVEDP (mmHg) 2.2 ± 0.5 11.9 ± 1.1 5.6 ± 0.5* 12.1 ± 0.9 11.8 ± 0.5 12.9 ± 1.3
dP/dt max (mmHg/s) 3559 ± 89 2503 ± 120 3054 ± 77* 2535 ± 100 2529 ± 61 2326 ± 99
dP/dt min (mmHg/s) 3022 ± 81 2004 ± 112 2549 ± 84* 2019 ± 107 1931.9 ± 55 1800 ± 89
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*

P<0.01 vs. other MI groups. MAP, mean arterial pressure; LVEDP, left ventricular end-diastolic pressure; dP/dtmax, maximum first derivative of pressure; dP/dtmin, minimun first derivative of pressure.

Intramyocardial injection of tissue kallikrein, but not inactive kallikrein, significantly reduced infarct size in the left ventricle 1 day after MI compared with the MI control group, as determined by TTC staining and quantitative analysis (Figure 1A and B). Co-administration of icatibant and L-NAME abrogated kallikrein’s effect. However, icatibant, L-NAME or aprotinin alone had no effect on myocardial infarct size as compared to the MI control (data not shown).

Figure 1.

Figure 1

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Tissue kallikrein administration reduces infarct size of kininogen-deficient BNK rats after MI, and the effect is blocked by icatibant and L-NAME; treatment with inactive tissue kallikrein had no effect. A) Representative heart sections stained with TTC. B) Quantitative analysis of infarct size. Infarct size is expressed as percentage of infarcted area to left ventricle area at risk. *P<0.01 vs. other groups, n=6–10.

Kallikrein Reduces MI-induced Cardiomyocyte Apoptosis and Intramyocardial Inflammation

Apoptotic cardiomyocytes were detected by TUNEL staining in the infarcted myocardium 1 day after MI, and kallikrein treatment reduced the number of apoptotic cells (Figure 2A). Icatibant and L-NAME abrogated kallikrein’s effect, and inactive kallikrein had no effect. Quantitative analysis showed that active, but not inactive, kallikrein significantly reduced the ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes as compared to the control group. However, kallikrein’s protective effect was blocked by icatibant and L-NAME (Figure 2B). Furthermore, inflammatory cell accumulation in the infarcted region of the heart was identified by ED-1 immunostaining (Figure 3A). ED-1 positive cells were counted for quantification of monocyte/macrophage number (Figure 3B). Increased inflammatory cell infiltration was detected in the infarcted area of the heart after acute MI, but kallikrein injection significantly decreased monocytes/macrophages compared with the control. Icatibant and L-NAME blocked kallikrein’s effect, but aprotinin-inactivated kallikrein had no protective effect against the inflammatory response.

Figure 2.

Figure 2

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Active tissue kallikrein inhibits cardiomyocyte apoptosis induced by MI in the infarcted heart of kininogen-deficient BNK rats, and the effect is blocked by icatibant and L-NAME; treatment with inactive tissue kallikrein had no effect. A) Representative apoptotic cells stained by TUNEL. B) Quantitative results of apoptotic cells. *P<0.05 vs. other groups, n=6–10.

Figure 3.

Figure 3

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Tissue kallikrein administration reduces monocyte/macrophage infiltration in heart tissue of kininogen-deficient BNK rats after MI, and the effect is blocked by icatibant and L-NAME; treatment with inactive tissue kallikrein had no effect. A) Representative monocyte/macrophage infiltration in infarcted heart tissue as determined by ED-1 immunohistochemical staining. B) Quantitative analysis of monocyte/macrophage number in infarcted heart after MI. *P<0.01 vs. other groups, n=6–10.

Kallikrein Increases NO Levels and Reduces Myeloperoxidase Activity

Kallikrein treatment resulted in a significant increase in cardiac NOx production compared with the MI control group, and this effect was abrogated by icatibant and L-NAME (Figure 4A). Moreover, kallikrein prevented the increase in cardiac myeloperoxidase levels induced by MI damage, and the effect of kallikrein was blocked by icatibant and L-NAME (Figure 4B). Again, inactive kallikrein had no effect in increasing NO formation or inhibiting oxidative stress.

Figure 4.

Figure 4

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Tissue kallikrein administration increases NO formation and decreases myeloperoxidase (MPO) activity in absence of kininogen after MI, and the effect is blocked by icatibant and L-NAME; treatment with inactive tissue kallikrein had no effect. A) NOx levels and B) MPO activity in infarcted heart tissue of kininogen-deficient BNK rats 1 day after MI. *P<0.01 vs. other groups, n=6–10.

Active Kallikrein Reduces Hypoxia/Reoxygenation (H/R)-induced Apoptosis and Caspase-3 Activity in Primary Cultured Cardiomyocytes

Representative Hoechst-positive staining and quantitative analysis showed that active tissue kallikrein, but not aprotinin-inactivated tissue kallikrein, significantly reduced H/R-induced apoptosis in cultured primary cardiomyocytes derived from BNK rats (Figure 5A and B). Similarly, active kallikrein, but not inactive kallikrein, significantly reduced H/R-induced caspase-3 activity (Figure 5C). Icatibant and L-NAME abrogated kallikrein’s effects on both apoptosis and caspase-3 activity.

Figure 5.

Figure 5

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Tissue kallikrein reduces apoptosis and caspase-3 activity in cultured cardiomyocytes derived from kininogen-deficient BNK rats after 12 hour-hypoxia followed by 24 hour-reoxygenation (H/R), and the effect is blocked by icatibant and L-NAME; treatment with inactive tissue kallikrein had no effect. A) Representative apoptotic cells stained by Hoechst. B) Quantitative analysis of apoptotic cells. C) Caspase-3 activity. *P<0.01 vs. other groups, n=3.

Discussion

This study establishes that tissue kallikrein elicits cardioprotection independent of kinin formation. Using kininogen-deficient BNK rats, which are unable to produce kinin peptides,7 we clearly demonstrated that tissue kallikrein has a direct role in protection against cardiac injury by reducing MI-induced infarct size, cardiomyocyte apoptosis and intramyocardial inflammation through kinin B2 receptor activation and NO formation. Similar results were observed from our previous studies using kininogen-expressing Wistar rats subjected to MI.14 More than two decades ago, we reported that tissue kallikrein rapidly induced rat uterine contraction despite kinin antiserum and kininase treatment.10 In addition, Erdös’ group8 reported that kallikrein can trigger kinin B2 receptor activation independent of kinin formation in cultured cells. Thus, our present study confirmed those previous studies. Moreover, ACE inhibition has been shown to potentiate kallikrein’s effect on kinin B2 receptor activation in the absence of kinin formation.9 Since ACE inhibitors are popular drugs used in the treatment of cardiovascular and related diseases, the current findings suggest that kallikrein may also contribute to the therapeutic effects of ACE inhibition via a kininogen/kinin-independent pathway. Taken together, these combined results indicate that tissue kallikrein has a direct function independent of its kinin-releasing activity in protection against cardiovascular disease. It is important to note that the involvement of local cardiac low molecular weight kininogens in BNK rats can be dismissed due to the finding that this rat strain possesses a point mutation in the kininogen gene that causes low molecular weight kininogen to accumulate inside the cell and thus prevent its secretion for cleavage by kallikrein.20 Moreover, rat T-kininogen should have no effect on our observations because it can not be cleaved by tissue kallikrein.21

It has been shown that only active tissue kallikrein, but not active site-inhibited kallikrein, can stimulate the kinin B2 receptor in cultured cells, suggesting that cleavage of a peptide bond in the receptor is necessary for its activation by kallikrein.8 To determine whether tissue kallikrein has a direct proteolytic action on the kinin B2 receptor, we evaluated the effect of purified active and inactive forms of tissue kallikrein on hypoxia/reoxygenation-induced programmed cell death in cultured cardiomyocytes. Our results showed that active kallikrein, but not inactive kallikrein, inhibited hypoxia-induced apoptosis and caspase-3 activity in cultured cardiomyocytes derived from Sprague-Dawley (data not shown) and kininogen-deficient rats. These results indeed indicate that cleavage of a peptide bond in the kinin B2 receptor is necessary for direct activation of kinin B2 receptor by tissue kallikrein.

Kinin is capable of generating NO by causing an increase in the phosphorylation of eNOS.22 Similarly, tissue kallikrein gene transfer has been shown to promote muscular neovascularization by eNOS upregulation and Akt activation.23 Moreover, eNOS gene delivery protected against cardiac remodeling through reduction of oxidative stress after MI.24 NO is a potent antioxidant25 and is capable of inhibiting neutrophil superoxide anion production via a direct action on the membrane components of NADPH oxidase and the assembly of NADH/NADPH oxidase subunits.26, 27 Our present finding showed that icatibant and L-NAME abolished kallikrein’s effects in promoting NOx (an indicator of NO) levels as well as suppressing superoxide production in infarcted hearts. These combined results indicate that kallikrein/kinin is capable of improving cardiac function through increased NO formation.

A recent study showed that the kinin B1 and B2 receptors may serve a protective role in cardiac dysfunction.28 However, we previously demonstrated that myocardial hypertrophy induced by aortic occlusion is mediated by B2 receptor, but not by B1 receptor, using kinin receptor knockout mice.29 In addition, intact bradykinin, but not des-Arg(9)-BK (a kinin B1 receptor agonist), prevented cardiomyocyte apoptosis and ventricular remodeling after acute ischemia/reperfusion, supporting a role of kinin B2 receptor, but not B1 receptor, in cardiac protection.3 The potential role of tissue kallikrein in protection against other organ damage such as the kidney, blood vessel and brain through direct kinin B2 receptor activation awaits further investigation.

It has been recently reported that the kallikrein-kinin system is involved in protease-activated receptor-mediated inflammation in rodents.30 In this regard, we observed that tissue kallikrein independent of kinin formation promotes the migration of cultured human keratinocytes via direct activation of protease-activated receptor-1 (unpublished results). Whether tissue kallikrein can act on protease-activated receptors to prevent ischemic heart disease, independent of its interaction with kinin receptors, is a new avenue for future studies.

Perspectives

This is the first study to demonstrate that tissue kallikrein improves cardiac function by inhibiting apoptosis and inflammation through direct activation of the kinin B2 receptor without kinin formation in the infarcted myocardium. Comparison of active and inactive tissue kallikrein indicates that cleavage of a peptide bond is required for B2 receptor stimulation by kallikrein in vitro and in vivo. This is an innovative finding as it is well characterized that tissue kallikrein exerts biological functions through generation of kinin peptides from kininogen substrate. Since ACE inhibition can potentiate kallikrein’s effect on kinin B2 receptor activation, tissue kallikrein, in addition to kinin formation, could provide advanced therapeutic benefits of ACE inhibition in protection against cardiovascular and renal diseases.

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health grants HL29397, DK066350, and C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources.

Footnotes

Disclosures

None.

References

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